Non-native polymerase encoding nucleic acid construct

ABSTRACT

The present invention provides an array of compositions useful for effecting and/or exhibiting changes in biological functioning and processing within cells and in biological systems containing such cells. In effect, these compositions combine chemical modifications and/or ligand additions with biological functions. The chemical modifications and/or ligand additions provide additional characteristics to the compositions without interfering substantially with their biological function. Such additional characteristics include nuclease resistance, targeting specific cells or specific cell receptors localizing to specific sites within cells and augmenting interactions between the compositions and target cells of interest as well as decreasing such interactions when desired. Also provided by the present invention are processes and kits.

FIELD OF THE INVENTION

[0001] This invention relates to compositions including nucleic acidconstructs, conjugates, and vectors which are capable of effecting andexhibiting biological function within a cell or cell containingbiological system.

[0002] All patents, patent publications, scientific articles cited oridentified in this application are hereby incorporated by reference intheir entirety in order to describe more fully the state of the art towhich the present invention pertains.

BACKGROUND OF THE INVENTION

[0003] An alternative to viral mediated gene delivery is direct deliveryof nucleic acid. This approach has several limitations including lowefficiency of transfer, low stability and lack of cell specificity. Inorder to overcome some of these limitations other approaches have beenmade. These include non-specific ionic complexes with polycations suchas polylysine (Wu and Wu, U.S. Pat. No. 5,166,320, contents of which areincorporated herein by reference) and histone. These bindnon-specifically with the nucleic acid construct through polycations orbasic proteins, such as histones. However, the resulting complexes stillsuffer some limitations including lack of uniformity of the complexes,lack of specificity with respect to polycation binding to specificregions of the nucleic acid construct, potential interference ofcomplexes with nucleic acid and possible untimely dissassociation of thecomplex or lack of timely disassociation of the complex leading to alack of stability of these nucleic acid polycation or nucleicpolypeptide complexes.

[0004] Nucleic acid transfer to cells can take place by various methods.Such methods can utilize free nucleic acid, nucleic acid constructs ornucleic acid as part of the genome of a virus or bacteriophage vector.

[0005] Wu et al., U.S. Pat. No. 5,166,320, utilized a polynucleotide ina nonspecific association with the polycation polylysine. Thesecomplexes suffer limitations including lack of consistency ofcomposition, lack of specificity with respect to polycation binding tospecific regions of the nucleic acid construct, potential interferenceof complexes with nucleic acid and possible untimely dissassociation ofthe complex or lack of timely disassociation of the complex leading to alack of stability of these nucleic acid or histone polycation or nucleicpolypeptide complexes. This procedure does not provide for delivery ofvirus vectors. Furthermore, cell transformation efficiencies are stilllow.

[0006] Methods for retrovirus mediated gene transfer to hematopoieticcells ex vivo has been attempted in the presence of fibronectin orfibronectin fragments. Fibronectin binds to retroviruses but not to anyother viruses, nucleic acids or nucleic acid constructs. Williams andPatel, WO 95/26200 (the contents of which are incorporated herein byreference), have transformed hematopoietic cells with retroviruses inthe presence of fibronectin. The use of fibronectin in this way islimited only to use with some retrovirus vectors and not with othervirus vectors or with nucleic acids.

[0007] It is desirable to form multimeric complexes for two primaryreasons. The formation of such complexes results in an additive effectsuch that one can obtain collective activity of the monomeric unitswithin a complex or these complexes could provide enhanced bindingproperties compared to the individual compounds or monomeric units,either through cooperative binding effects or through neighboringeffects which produce higher localized concentrations. Polyligandsusually exhibit higher binding affinities in the polymeric form than inthe monomeric form as seen by the binding of polynucleotide sequences totheir complementary sequences when compared to the binding of themonomeric units.

[0008] Multimeric complexes have been formed either by crosslinking ofmonomeric compounds directly or through a matrix or through theformation of noncovalent linkages. Examples of multimeric complexesformed by the crosslinking of a given compound such as enzymes, eitherdirectly or through a matrix are described in U.S. Pat. No. 4,687,732(contents of which are incorporated herein by reference), whereby avisualization polymer composed of multiple units of a visualizationmonomer is linked together covalently by coupling agents which bond tochemical groups of the monomer. Examples of multimeric complexes madethrough the formation of noncovalent linkages such as ligand-receptorsystems are the PAP (peroxidase-anti-peroxidase) complexes and APAAP(alkaline phosphatase-anti-alkaline phosphatase) complexes in common useas immunological reagents and the streptavidin-biotinylated enzymecomplexes used for detection of biotinylated entities.

[0009] In the case of complexes formed by crosslinking or noncovalentbinding, there are limitations with respect to the spacing and thechemical milieu of the monomeric unit within the complex which mayaffect the function and activity of the monomeric unit and as the sizeof the complex grows, solubility may be affected.

[0010] Efforts to regulate expression of procaryotic genes by eucaryoticprocesses have been attempted by Schwartz et al. (1993 Gene 127: 233)(also incorporated herein by reference) who introduced an intronsequence from a eucaryotic gene into a procaryotic gene. However, whenintroduced into a cell capable of mRNA processing, the gene expressed analtered protein in which additional amino acids were present due to thepresence of flanking exon sequences associated with the inserted intron.This limitation is inherent in this approach since this method of intronisolation requires the a priori presence of inherent restriction sitesin the exon regions flanking the intron, and intron insertion requiresthe presence of appropriate restriction sites in the gene receiving theintron. Therefore, even after the excision of the intron from the RNA,the flanking exon sequences remain as part of the coding sequence of themature RNA molecules. Furthermore, the number of sites for introninsertion on the receiving gene is severely limited by the availabilityof appropriate restriction sites.

[0011] The alteration of the gene product by this approach may haveunpredictable effects on the function of the gene product and severelylimits the applicability of this method to specific instances. In theexample of Schwartz et al. the additonal amino acids had no apparenteffect on the activity of the protein synthesized in the capable cell,but this is not always a predictable quality since it depends upon thesite where the additional amino acids are incorporated. For instance, ashort sequence coding for a small peptide introduced into the amino endof T7 RNA polymerase by Dunn et al. (1988 Gene 68: 259) (alsoincorporated herein by reference) had no apparent effect on enzymeactivity. However introduction of the same sequence into a site near thecarboxy terminus resulted in nearly complete loss of enzyme activity.Thus, the incorporation of extra amino acids as a result of introducingan exon into a coding sequence by the method of Schwartz et al. couldhave a drastic mutagenic effect.

[0012] Systems derived from procaryotic elements can produce functionalproducts in mammalian cells. T7 RNA polymerase, an enzyme derived froman E coli bacteriophage, has been expressed both transiently and stablyin mammalian systems (Fuerst et al., 1986 Proc. Nat. Acad. Sci. U.S.A.83: 8122, the contents of which are herein incorporated by reference).When synthesized in a mammalian environment, it is capable of actingupon genes under the control of a T7 promoter to produce transcriptsthat can be translated to provide a functional gene product. Largeamounts of RNA can be transcribed from the T7 promoter (comprising up to30,000 RNA molecules per cell, Lieber et al. 1993, also incorporated byreference).

[0013] In eucaryotic systems success has only been achieved by the useof a binary system with the polymerase on one construct and the T7promoter on a separate construct, In this way either sequentialtransfections (Lieber et al. 1989 Nucleic Acids Res 17, 8485) (alsoincorporated by reference) or co-transfections with separate plasmids(Lieber et al. 1993 Methods Enzym. 217, 47) (incorporated by reference)or transfection with a plasmid containing a T7 promoter followed byinfection with a recombinant vaccinia virus coding for T7 RNA polymerase(Fuerst et al. 1986 Proc. Nat. Acad. Sci. U.S.A. 83, 8122) must be done.Since T7 RNA polymerase can be cloned only free of a T7 promotersequence (Davenloo et al. 1984 Proc. Nat. Acad. Sci. U.S.A. 81: 2035)(incorporated herein by reference), it appears that attempts to cloneboth elements in a single construct fail due to an event where synthesisof the T7 RNA polymerase-initiated transcription from the downstreampromoter continues around the plasmid to direct more synthesis of T7 RNApolymerase leading to a cytocidal autocatalytic cascade. A similarstrategy of elimination of cognate promoters has been described for thecloning of the bacterophage T3 (Morris et al. 1986 Gene 41: 193) and SP6(Kotani et al. 1987 Nucl. Acids Res. 15: 2653) (both publicationsincorporated herein by reference) RNA polymerases. However,compatibility of these elements has been achieved by the addition of twomodifications to the construct, i.e., inhibition of the T7 RNApolymerase by the presence of T7 lysozyme and the use of a repressibleT7 lac promoter (Dubendorff and Studier 1991 J. Mol. Biol. 219: 61,1991, incorporated herein by reference). Both of these limitations arerequired in order to obtain a construct.

[0014] The introduction of genetic material into cells can be done bytwo methods. One method is the exogenous application of nucleic acidswhich act directly on cellular processes but which themselves are unableto replicate or produce any nucleic acid. The intracellularconcentrations of these molecules that must be achieved in order toaffect cellular processes is dependent on the exogenous supply. Anothermethod for nucleic acid delivery is the introduction into cells ofPrimary Nucleic Acid Constructs which themselves do not act on cellularprocesses but which produce single stranded nucleic acid in the cellwhich acts on cellular processes. In this case the introduced PrimaryNucleic Acid Construct can integrate into cellular nucleic acid or itcan exist in an extrachromosomal state, and it can propagate copies ofitself in either the integrated or the extrachromosomal state. Thenucleic acid consstruct can produce, from promoter sequences in thePrimary Nucleic Acid Construct, single stranded nucleic acids whichaffect cellular processes of gene expression and gene replication. Suchnucleic acids include antisense nucleic acids, sense nucleic acids andtranscripts that can be translated into protein. The intracellularconcentrantions of such nucleic acids are limited to promoter-dependentsynthesis.

[0015] The effectiveness of single stranded nucleic acids produced fromprimary nucleic acid constructs is dependent on their concentration, thestability and the duration of production in the cell. Current methodsfor achieving intracellular concentrations are limited by a dependenceon promoter directed synthesis.

[0016] The effectiveness of antisense therapy depends depends in largepart on three major factors: a) the rate of transcription of antisenseRNA, b) the cellular location of the RNA and c) the stability of the RNAmolecules. While previous studies have addressed each of these factors,all three have not been addressed in a single approach. The presentinvention utilizes AS sequences substituted for nucleotide sequences inthe U1 and other hnRNAs to achieve high nuclear concentrations of stableantisense RNA sequences.

[0017] U1, U2 and other snRNAs are nuclear-localized RNA moleculescomplexed with protein molecules. (Dahlberg and Lund 1988 in Structureand Function of Major and Minor Small Nuclear RibonucleoproteinParticles, M. Birnstiel, Ed., Springer Verlag, Heidelberg, p38: , Zieveand Sautereau 1990 Biochemistry and Molecular Biology 25;1, all of whichare incorporated herein by reference).

[0018] The various promoters for U1, U2 and other snRNA operons are verystrong and produce large amounts of RNA. U1 and other snRNAs havesignals for export to the cytoplasm where specific proteins arecomplexed before reimportation to the cytoplasm as snRNPs (FIG. 41).snRNAs are very stable molecules. They form very highly ordered stem andloop structures (FIG. 43) which, when complexed with specific proteins,form snRNP, or splicesomes.

[0019] Antisense and other nucleic acid molecules which affect geneexpression by acting on and altering RNA transcripts can derive certainadvantages by confinement to the nucleus. Higher concentrations can bemaintained in the smaller volume of the nucleus, interactions withtarget RNA can occur prior to their being used for expression and therewould be no competition with messenger binding ribosomes.

[0020] Addition of antisense sequence to U2 RNA (Izant and Sardelli 1988in Current Communications in Molecular Biology, Cold Spring Harbor,p141, incorporated herein by reference) as a means of deliveringantisense sequences altered the properties of normal U2 transcripts.Hybrid U2 molecules formed by insertion of antisense sequences into arestriction site in the 5′ end of the U2 transcript region showeddecreasing antisense effectiveness with increasing insert size. Insertslonger than 250 bases substantially reduced antisense effectiveness.Furthermore, hybrids did not accumulate in the nucleus as efficiently astheir wild type counterparts with the fraction of hybrids in the nucleusdecreasing as insert length increased.

[0021] Yu and Weiner (1988 in Current Communications in MolecularBiology, Cold Spring Harbor, p141, contents incorporated by reference)substituted 9 base antisense sequences directed at target sequencessurrounding splice sites in mRNA. The antisense substitutions were madeat the 5′ end of U1 RNA. None of the antisense substitutions affectedthe level of targeted species of mature cytoplasmic RNA.

[0022] Constructs have been designed to increase antisense effectivenessby the inclusion of more than one targeting element in a singletranscriptional unit. Multivalent constructs prepared in this way canproduce numerous target directed entities acting on multiple targetsites in nucleic acids. (Chen et al. 1992 in Antisense Strategies,Annals of the New York Academy of Sciences 660;271: Zhow et al. Gene1994 149;33, both publications incorporated herein by reference).Different approaches to inhibition can be incorporated into amultivalent transcript as shown by Lisziewicz et al. (1993 Proc NatlAcad Sci USA 90, 8000, also incorporated by reference) who combinedmultiple copies of the HIV TAR with an antisense sequence to HIV gag onthe same transcript.

[0023] The use of multivalent targeting by the inclusion of more thanone targeting element on the same transcript provides a a method forcounteracting the the high mutation rate of viruses such as HIV due tothe unlikely event of simultaneous mutation of multiple targetsequences. However, the common means of accomplishing these designs isthe inclusion of the product entities on a single transcript. Thisapproach suffers from the following limitations:

[0024] a) The total number of RNA molecules available as effectiveentities is limited by the strength of the single promoter;

[0025] b) During stable transformation of a cell, the integration eventcan disrupt the nucleic acid template sequence responsible forexpression of the antisense sequence;

[0026] c) The use of multivalent transcripts is not favorable when oneproduct entity present on the transcript acts on targets present in onecellular locale and another product entity present on the samemultivalent transcript acts on targets present in a different cellularlocale. This was the approach reported by Lisziewicz et al. (1993) wheremultiple TAR sequences, which act to bind the HIV tat protein in thecytoplasm, were present on the same transcript with antisense sequencesfor the HIV gag RNA, which are most effective in the nucleus.

[0027] Although there have been major efforts to find effectiveantiviral treatments, at the present time the only success has been in adimunition of virus growth rather than elimination of the virus. Amongthe efforts that have been pursued are attempts to prevent initiation ofthe virus replication cycle by preventing the virus from entering thecell by immunization or by treatment with antibodies or with proteinsthat interfere with virus recognition of a cell by interacting with thevirus or the virus receptor site on the cell. These include unsuccessfultreatment with high levels of soluble CD4 (Husson et al., 1992,incorporated by reference). In addition, efforts have been made tocombat HIV infection after virus entry into a cell using proteaseinhibitors for preventing processing of viral polypeptides intofunctional proteins and varied nucleoside analogues which can blockreplication of the virus by inhibiting the activity of the virallyencoded reverse transcriptase and other functions necessary for viruspropagation. Stages of the processes of viral infection and viralreplication cycle have been examined for the possibility ofpharmacological or immunological intervention of the disease process.However, as independent and effective therapeutic agents, bothimmunological and small molecule inhibitors have failed to stem theprogression of AIDS, and major problems remain in terms of effectivenessand the rise of viruses resistant to small molecule therapeutic agents.Even the application of combinations of immunological and small moleculeagents has not been successful.

[0028] The introduction of genetic information into cells either toreplace a function or to introduce a new function has provided aneffective means for the treatment of viral infection. Genetic therapyapproaches have been used to impart cell resistance to viruses bymechanisms which act intracellularly on the viral replication process(see Yu et al., Gene Therapy 1, 13-16 [1994, incorporated by reference).A result of these studies is that, in vitro, the effectiveness ofgenetic therapies is sensivitive to virus concentration. Experiments invitro that showed substantial levels of resistance at low ratios ofvirus to cells, at higher ratios showed a “breakthrough” phenomenoncharacterized by a period of seeming effectiveness followed by a surgein the virus production (Sczakiel et al. 1992 J. Virol 66;5576 :Scakieland Pawlita 1990 J. Virol. 65;468, all of which are incorporated byreference). Thus in vitro, at lower virus:cell ratios some geneticallytreated cells demonstrate longer survival times that at highervirus:cell ratios.

[0029] Compartmentalization of function is critical to regulatedprocesses in eucaryotic cells. For example, the major part of cellularDNA is organized into chromosomes located in the nucleus wheretranscription of genetic information takes place. The major part of RNAsynthesized in the nucleus is transported to the cytoplasm where it istranslated. Other subcellular compartments for localized functioninclude the Golgi apparatus, endoplasmic reticulum, nucleolus,mitochondria, chloroplast and the cellular membrane. Thus, a variety ofmechanisms exist either to retain macromolecules in specific cellularcompartments or to transport macromolecules from one cellularcompartment to another. For example, in the directed exit of mature mRNAout of the nucleus into the cytoplasm, the presence of a 5′ cap, removalof introns and addition of a poly A sequence are all believed tocontribute to the signal that directs the relocation (reference).

[0030] Some RNAs, such as small nuclear RNAs (snRNAs) involved insplicesome assembly, are relocated by sequential transportation(Dahlberg and Lund, 1988, in Structure and Function of Major and MinorSmall Ribonuclear Particles, M. Birnstiel, ed., Springer Veriag,Heidelberg, pg. 38, incorporated by reference ). After transcription inthe nucleus, the presence of the 5′ cap and the processed 3′ terminusgenerate a bipartite signal for transport of U1 RNA into the cytoplasm.At this point there is further processing of the RNA by excision of afew nucleotides and hypermethylation of the 5′ cap. The binding ofsplicesome proteins present in the cytoplasm to the Sm region of the U1RNA in combination with the hypermethylation is believed to generate asignal for the reimportation of the RNA back into the nucleus.

[0031] In contrast to most mRNA, most proteins do not need to betransported from their site of synthesis in the cytoplasm. However, someproteins that function in transcription, replication or other nuclearmaintenance functions need to be present in the nucleus to functionproperly. In this case a polypeptide signal sequence present in theprotein directs the transport of the protein from the cytoplasm into thenucleus. Still other proteins are not functional in the cytoplasm or inthe nucleus but are required to be present in the membrane of the cellthereby requiring the presence of leader and lipophilic sequences.

[0032] The directing of target molecules as an approach to genetictherapy has been studied by attempts at localization for the expresspurpose of putting an active agent such as antisense RNA in proximity tothe target in a particular cellular locale For example, some workershave designed nucleic acid constructs to express anti-sense RNA thatwould be retained in the nucleus in order to block newly transcribedtarget RNA from functioning (Izant and Sardelli, 1988, CurrentCommunications in Molecular Biology, D. Melton, ed., Cold Spring HarborLaboratory; Cotten and Birnstiel, 1989, EMBO Journal 8, 3861,incorporated by reference). The opposite effect has also been achievedby designing the transcript to include a signal for enhancing transportinto the cytoplasm in order to block the translation of RNA that may bepresent there (Liszeiwicz et al. 1993, incorporated by reference).

SUMMARY OF THE INVENTION

[0033] The present invention overcomes the above-described limitationsin the prior art by providing compositions which retain their biologicalfunction within cells or biological systems containing such cells uponchemical modification which may add further useful biological functionsin addition to those which are retained.

[0034] The present invention relates to nucleic acid constructs capableof biological function and processing within a cell. These constructsmay contain chemically modified biological or synthetic compounds. Theseconstructs retain their biological function within a cell, but may alsobe able to exhibit additional properties by virtue of the chemicalmodification. The constructs combine chemical modifications andbiological functions integrated within the construct.

[0035] The invention relates to novel constructs that have eitherincorporated unique biological elements or have incorporated chemicalentities that introduce new properties to the construct, or both.

[0036] Unique biological elements are either synthetic, non-nativeheterologous or artifical elements in the construct that when in thecell provide novel capabilities (non-native) or novel products(artificial). Novel capabilities are provided by but are not limited tothe introduction of such elements as heterologous processing elementsthat allow the construct to function in compatable cells, signalingelements for localization within the cell and multi-independentproduction cassettes.

[0037] Chemical modifications provide added characteristics to theconstructs without interferring substantially with its biologicalfunction. Such added characteristics can be, but are not limited tonuclease resistance, the capability of targeting specific cells orspecific receptors on cells, the capability of localization to specificsites within a cell, or the ability to enhance the interaction betweenthe construct (or virus or vector) and the target cell in a generalmanner or too prevent or interfere with such interaction when desired.

[0038] The invention combines biological elements and chemicalmodification either to create a construct that defines its function, itslocation within a cell and its fate, or to modulate the interaction ofthe virus, vector or construct and cell prior to the entry of the virus,vector or construct into the cell.

[0039] Furthermore, the present invention relates to methods andconstructs that provide for general interactions between target cellsand a nucleic acid entity and compositions of multimeric complexesuseful in vivo and in vitro.

[0040] Among the compositions provided by this invention is a constructwhich when present in a cell produces a product. The construct comprisesat least one modified nucleotide, a nucleotide analog or a non-nucleicacid entity, or a combination of any of the foregoing. Anothercomposition is a construct bound non-ionically to an entity comprising achemical modification or a ligand. When present in a cell, such aconstruct produces a product. Another composition provided by thisinvention is a construct bound non-ionically to an entity comprising achemical modification or a ligand. When present in a cell, such aconstruct also produces a product.

[0041] This invention provides a composition comprising (a) anon-natural entity which comprises at least one domain to a nucleic acidcomponent; and at least one domain to a cell of interest; andoptionally, (b) the nucleic acid component, and optionally, (c) the cellof interest, or both (b) and (c). In this composition, the domain ordomains to the nucleic acid component are different from the domain ordomains to the cell. A kit is provided for introducing a nucleic acidcomponent into a cell of interest. This kit comprises in packagedcontainers or combination one element and three optional elements. Thefirst element is a non-natural entity which comprises at least onedomain to the nucleic acid component, and a domain to the cell ofinterest. Optional elements include the nucleic acid component, the cellof interest, and buffers and instructions.

[0042] Another composition provided by this invention comprises anentity which comprises at least one domain to a cell of interest, suchdomain or domains being attached to a nucleic acid component which is innon-double stranded form. A kit is also provided for introducing anucleic acid component into a cell of interest. The kit comprises inpackaged containers or combination an entity which comprises a domain tothe cell of interest, the domain being attached to a nucleic acidcomponent which is in non-double stranded form. Optionally provided arebuffers and instructions.

[0043] Also provided is a composition comprising an entity whichcomprises a domain to a nucleic acid component, the domain beingattached to a cell of interest. A kit is provided for introducing anucleic acid component into a cell of interest in packaged containers orcombination, the kit comprises an entity which comprises a domain to thenucleic acid component, the domain being attached to the cell ofinterest. Buffers and instructions may also be optionally included.

[0044] Further provided is a multimeric complex composition comprisingmore than one monomeric unit attached by means of one or moreinteractions. Thus, the monomeric units may be attached to each otherthrough polymeric interactions, or to a binding matrix through polymericinteractions, or a combination of both kinds of interactions.

[0045] Also provided is a multimeric composition comprising more thanone component attached to a charged polymer. In this composition, thecharged polymer is selected from a polycationic polymer, a polyionicpolymer, a polynucleotide, a modified polynucleotide and apolynucleotide analog, or any combination of the foregoing elements.

[0046] The present invention provides a nucleic acid construct whichwhen introduced into a cell codes for and expresses a non-nativepolymerase. The non-native polymerase is capable of producing more thanone copy of a nucleic acid sequence from the construct. Also provided isa nucleic acid construct which when introduced into a cell produces anucleic acid product comprising a non-native processing element. Whencontained in a compatible cell, the processing element is substantiallyremoved during processing.

[0047] This invention also provides a process for selectively expressinga nucleic acid product in a cell, the product requires processing forfunctioning. The process comprises first, providing a nucleic acidconstruct which when introduced into a cell produces a nucleic acidproduct comprising a non-native processing element, which issubstantially removed during processing when contained in a compatiblecell, and second, introducing the construct into the cell.

[0048] Another composition comprises a primary nucleic acid componentwhich upon introduction into a cell produces a secondary nucleic acidcomponent which is capable of producing a nucleic acid product, or atertiary nucleic acid component, or both. Neither the secondary nucleicacid component, the tertiary nucleic acid component, nor the nucleicacid product are capable of producing the primary nucleic acidcomponent.

[0049] Also provided herein is a process for localizing a nucleic acidproduct in a eukaryotic cell. This localizing process comprises first,providing a composition of matter comprising a nucleic acid componentwhich when present in a cell produces a non-natural nucleic acidproduct. The non-natural nucleic acid product comprises a portion of alocalizing entity, and a nucleic acid sequence of interest. In thesecond step of the process, the composition is introduced into aeukaryotic cell or into a biological system containing a eukaryoticcell.

[0050] Additionally provided by this invention is a nucleic acidcomponent which upon introduction into a cell is capable of producingmore than one specific nucleic acid sequence. Each such specificsequence so produced is substantially nonhomologous with each other andare either complementary with a specific portion of a single-strandednucleic acid of interest in a cell or capable of binding to a specificprotein of interest in a cell.

[0051] This invention further provides a process for increasing cellularresistance to a virus of interest. The process comprises first,providing transformed cells phenotypically resistant to the virus; and areagent capable of binding to the virus or to a virus-specific site onthe cells. Second, the process comprises administering theaforementioned reagent to a biological system containing the cells toincrease the resistance of the cells to the virus of interest.

[0052] Further provided is a nucleic acid construct which whenintroduced into a cell produces a non-natural product. The non-naturalproduct comprises two components: first, a binding component capable ofbinding to a cellular component, and second, a localization componentcapable of dislocating the cellular component when it is bound to thenon-natural product.

[0053] Also contemplated by the present invention is a process fordislocating a cellular component in a cell. Here, the process comprises,comprises first, providing a nucleic acid construct which whenintroduced into a cell produces a non-natural product, the productitself comprising two components: a binding component capable of bindingto a cellular component, and a localization component capable ofdislocating the cellular component when it is bound to the non-naturalproduct. In the second step of the process, the nucleic acid constructis introduced into a cell of interest or a biological system containingthe cell or cells of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054]FIG. 1 depicts the localized attachment of ligands and othermoieties to a nucleic acid construct by incorporation into a nucleicacid primer.

[0055]FIG. 2 depicts the dispersed attachment of ligands to a nucleicacid construct by extension from a modified nucleic acid primer.

[0056]FIG. 3 illustrates the dispersed attachment of ligands to anucleic acid construct by synthesis of a complementary RNA strand thatutilizes modified ribonucleotide precursors.

[0057]FIG. 4 illustrates the localized attachment with a nucteic acidconstruct by hybridization of a gapped circle with a modified nucleicacid moiety that also contains useful moieties incorporated into a 3′tail.

[0058]FIG. 5 illustrates the preparation of a gapped circle such asshown in FIG. 4.

[0059]FIG. 6 illustrates the localized attachment with a nucleic acidconstruct by hybridization of a gapped circle with a modified nucleicacid moeity with an unmodified 3′ tail to which has been hybridized anucleic acid with useful ligands incorporated thereinto.

[0060] FIGS. 7 AND 8 show the process for introducing a segment of RNAinto a cell by means of a modified primer whereby the RNA will betransformed in vivo into a double-stranded DNA segment.

[0061] FIGS. 9 AND 10 show the process for introducing a segment of RNAinto a cell by means of modified primers whereby the RNA will betransformed in vivo into double-stranded DNA segments.

[0062]FIG. 11 illustrates a process for introducing a segment of singlestranded DNA having modified nucleotides as part of its sequence.

[0063]FIG. 12 illustrates the fate of the modified single-stranded DNAfrom FIG. 11 after it has been introduced into a cell.

[0064]FIG. 13 illustrates a process for introducing a segment of doublestranded DNA having modified nucleotides as part of the sequence on eachstrand.

[0065]FIG. 14 illustrates a divalent antibody binder with one portionhaving an affinity for binding a retroviral particle, and the otherportion having an affinity for binding the CD34 antigen.

[0066]FIG. 15 shows the covalent attachment of DNA to each portion of anF(ab′)₂ antibody fragment with an affinity for the CD34 antigen.

[0067]FIG. 16(A) depicts the covalent attachment of DNA to an adenovirusbinding portion of a divalent antibody in order to promote the bindingof an AAV vector DNA molecule to a CD34 receptor.

[0068]FIG. 16(B) is the same depiction as in FIG. 16(A) except thatF(ab′) fragments are used instead of complete antibody proteins.

[0069]FIG. 17 illustrates a monovalent antibody to an adenovirus spikeprotein with one portion being modified by covalent attachment of DNAthat can bind an adenovirus associated virus (AAV) vector DNA moleculethrough hybridization and the other portion being modified by thecovalent attachment of an oligolysine modified by the attachment oflactyl groups.

[0070]FIG. 18 shows a monovalent antibody to an adenovirus spike proteinin which each portion of the antibody has been modified by the covalentattachment of lactosylated DNA molecules which are bound to an AAVvector DNA by means of hybridization.

[0071] FIGS. 19 AND 20 describe the synthetic steps for producing areagent that is useful for attaching nucleic acid moieties to anantibody.

[0072]FIG. 21 depicts a process for mutlimerization of F(ab′)₂ antibodyfragments by hybridization of nucleic acid homopolymers.

[0073]FIG. 22 depicts a process for multimerization of insulin moleculesby hybridization of nucleic acid homopolymers.

[0074]FIG. 23 depicts a process for multimerization of insulin moleculesby hybridization of nucleic acid heteropolymers with a binding matrix.

[0075]FIG. 24 shows the introduction of an SV40 intron sequence thatreconstitutes appropriate signals for in vivo splicing and production ofa normal mRNA transcript for T7 RNA polymerase.

[0076]FIG. 25 shows the process of the intron introduction andsubsequent construction of a T7 expression vector.

[0077]FIG. 26 shows the oligomers and their products used for thesynthesis of the SV40 intron containing T7 RNA polymerase codingsequence.

[0078]FIG. 27 depicts the process for the introduction of nucleotidesequences for the nuclear localization signal.

[0079]FIG. 28 is a comparison of the 5′ ends of the nucleotide sequencefor the normal T7 RNA polymerase and a T7 RNA polymerase with sequencesinserted for a nuclear localization signal.

[0080]FIG. 29 shows the process for the assembly of PCR generatedfragments by cloning methods to assemble a clone that directs thesynthesis of an intron containing T7 RNA polymerase transcript.

[0081]FIG. 30 shows the sequences for HIV antisense sequences and theprocess for their cloning into T7 directed transcription units.

[0082]FIG. 31 shows the cloning steps for the combination of T7 directedantisense into a clone that contains the intron containing T7 RNA polymerase.

[0083]FIG. 32 shows the DNA sequences and subsequent cloning steps formaking a protein expression vector.

[0084]FIG. 33 shows a process for a combination of the polylinkersequence from FIG. 32 and a T7 promoter and a T7 terminator for making aT7 directed protein expression vector.

[0085] FIGS. 34 AND 35 depicts the design of a primary nucleic acidconstruct that will function as a production center to generate singlestranded antisense DNA.

[0086]FIG. 36 depicts the design of a primary nucleic acid constructthat will generate a secondary nucleic acid construct capable ofdirecting transcription.

[0087] FIGS. 37 AND 38 depict the design of a primary nucleic acidconstruct that will generate a double hairpin production center(secondary nucleic acid construct).

[0088]FIG. 39 depicts the design of a primary nucleic acid constructthat will generate a production center (secondary nucleic acidconstruct) capable of inducible suicide.

[0089]FIG. 40 depicts the design of a primary nucleic acid constructthat will use tRNA primers in vivo to make secondary nucleic acidconstructs capable of transcription.

[0090]FIG. 41 depicts the process of excision of normal sequences from aU1 transcript region and replacement with novel sequences.

[0091]FIG. 42 shows the oligomer sequences for making HIV antisensesequences and the insertion of these oligomers as replacement for aportion of the U1 transcript sequence in a clone containing a U1 operon.

[0092]FIG. 43 is a computer generated secondary structure prediction forU1 transcripts with HIV antisense sequence substitutions.

[0093]FIG. 44 depicts the cloning process for making of a clone thatcontains multiple HIV antisense containing U1 operons.

[0094]FIG. 45 depicts the cloning steps for constructing a clone thatcontains multiple independent HIV antisense containing T7 directedtranscripts.

[0095]FIG. 46 shows the final structures of the multiple operonconstructs described in FIGS. 44 and 45.

[0096]FIG. 47 depicts the cloning steps for insertion of multiple T7antisense operons into a vector coding for the T7 intron containing RNApolymerase

[0097]FIG. 48 represents flow cytometry data measuring binding ofanti-CD4+antibody to HIV resistant U937 cells.

[0098]FIG. 49 shows PCR amplification of the gag region indicating theabsence of HIV in viral resistant cell line (2.10.16) after challenge.

[0099]FIG. 50 depicts a model system for testing the potentialinhibition of HIV antisense sequences by using beta-galactosidaseactivity as an indicator.

[0100]FIG. 51 is a table of data demonstrating the effect of the HIVantisense sequence upon beta-galactosidase activity by enzyme assays aswell as in situ assays.

DETAILED DESCRIPTION OF THE INVENTION

[0101] Definitions

[0102] Some definitions for the terminology used in the art and/or inthe present invention might be in order.

[0103] Primary Nucleic Acid Construct. A composition consisting ofnucleic acid which in a cell propagates Production Centers.

[0104] Production Center. A nucleic acid molecule derived from a PrimaryNucleic Acid Construct which in a cell is able to propagate otherProduction Centers or to produce single stranded nucleic acid.

[0105] Propagation. The generation or formation of a Production Centerfrom a Primary Nucleic Acid Construct or the generation or formation ofa Production Center from another Production Center.

[0106] Production. The generation of a single stranded nucleic moleculesfrom a Production Center.

[0107] Inherent Cellular Systems. Cellular processes and componentspresent in cells which can be utilized for the Production andPropagation as well as the function of single stranded Nucleic AcidProducts. Such processes and components can be native to the cell, or beintroduced to the cell by artificial means or by infection by, forexample, a virus.

[0108] 1. Gene Transfer Mediated by Ligands

[0109] The present invention is a defined chemically modified nucleicacid construct (CHENAC) which, upon introduction into a cell, is capableof biological function, i.e., production of a nucleic acid, productionof a protein in a cell or interaction with a nucleic acid or protein ina cell. The said chemical modification directly or indirectly rendersthe construct capable of one or more of the following properties: a)binding to a target cell b) nuclease resistance c) providing a mechanismfor introduction of the nucleic acid into cells d) providing nucleaseresistance within the cytoplasm e) facilitating transfer of the nucleicacid from the cytoplasm to the nucleus f) providing a longer lifetimewithin the cell g) providing a signal for integration into cellular DNA.In the present invention, one or more of the above properties is capableof being provided without substantially interfering with the biologicalfunction of said nucleic acid. The present invention uses chemicalmodification of nucleic acid to attach directly or indirectly one ormore ligands or chemical modifications or other moieties to a nucleicacid construct. A construct modified by the addition of ligands orchemical modifications could further complex with other moieties, thosemoieties being natural or unnatural, modified or unmodified oligo- orpolypeptides; polycations; natural or unnatural, modified or unmodifiedoligo- or polysaccharides; multimolecular complexes; inactivatedviruses; and any chemical binding, attachment or conjugation capable ofcomplexing with the ligand or chemical moiety. The Modified Nucleic AcidConstructs of the present invention provide for the delivery of nucleicacid to eucaryotic cells including the cells of plants, humans and othermammals and to procaryotic cells.

[0110] The present invention provides the capability to localizechemical modifications to regions of the CHENAC. This permitsconstruction of compositions in which the segment of the CHENACresponsible for the biological function can be segregated from modifiedregion(s) responsible for the properties listed above in cases where theaddition of ligands or chemical modifications could be disruptive tobiological function. In cases where ligands or chemical modificationscan interfere with biological activity, chemically modified segments ofthe CHENAC could be segregated from the construct subsequent tointroduction into the cell by displacement or loss of modified segments.

[0111] In one aspect, this invention provides a construct which whenpresent in a cell produces a product, the construct comprising at leastone modified nucleotide, a nucleotide analog and a non-nucleic acidentity, or a combination of the foregoing. The modified nucleotide maybe chemically modified as described further below. When present in theconstruct, at least one of the nucleotide analog or analogs may also bemodified either on the backbone or the side chain or on both positions.With respect to the non-nucleic acid entity this element may also beattached to a single strand or both strands of the construct when thelatter is double stranded.

[0112] The non-nucleic acid entity or entities may take any number ofdiverse forms. These include natural polymers, synthetic polymers,natural ligands and synthetic ligands, as well as combinations of anyand all of the foregoing. When the non-nucleic acid entity or entitiestake the form of a natural polymer, suitable members may be modified orunmodified. Natural polymers can be selected from a polypeptide, aprotein, a polysaccharide, a fatty acid, and a fatty acid ester as wellas any and all combinations of the foregoing.

[0113] When the present invention contemplates the use of a syntheticpolymer for the non-nucleic acid entity or entities, homopolymers andheteropolymers may be employed. Such homopolymers and heteropolymers arein many ways preferred when they carry a net negative charge or a netpositive charge.

[0114] It is significant that the above-described construct of thepresent invention can be designed to exhibit a further and additionalbiological activity which is usefully imparted by incorporating at leastone or more modified nucleotides, nucleotide analogs, nucleic acidentities, ligands or a combination of any or all of these. Suchbiological activity may itself take a number of forms, includingnuclease resistance, cell recognition, cell binding, and cellular(citoplasmic) or nuclear localization.

[0115] The nucleic acid of the CHENAC can be DNA, RNA, a combination ofRNA and DNA, e.g., a DNA-RNA hybrid or a chimeric nucleic acid, such asa DNA-RNA chimera. The nucleic acid components of the CHENAC can besingle-stranded or double-stranded. The nucleic acid component can beall or in part a modified nucleic acid or a nucleic acid analogue.Modified nucleic acids are polymers capable of binding to complementaryregions of nuiceic acids and which contain chemical modifications of thesugar, base or phosphate moieties.

[0116] Nucleic acid analogues are polymers capable of binding to acomplementary nucleic acid and in which these polymer backbones areother than ribo and deoxyribose sugars and phosphate groups or in whichside chain groups are other than natural or modified bases. Examples ofnucleic acid analogue polymers include peptide nucleic acids or whichhave side chains containing such non-discriminatory base analogues, orunivesal bases, as 1-(2′-deoxy-β-D-nbofuranosyl)-3-nitropyrrole (Nicholset al. 1994 Nature 369;492,) or 2′-deoxynebularine and2′-deoxyxanthosine (Eritja et al. 1986 Nucleic Acids Research 14;8135),both publications being incorporated herein by reference.

[0117] Modified nucleic acids, nucleic acid analogues and other polymerswith a net negative charge and/or a functional amino group(s) mayfacilitate the practice of this invention, since these propertiesprovide for solubility, specificity, enzyme function and binding. It maybe preferred that some of the functional sequences of nucteic acid maybe natural or modified nucleic acid sequences such as promotersequences, terminator sequences or priming binding sequences.

[0118] The nucleic acid component of the CHENAC can be single stranded,double stranded, partially double stranded or even triple stranded.Further, such component can be circular or linear or branched, and maytake the form of any DNA or RNA. It can contain both double stranded andsingle stranded regions and it can contain an non-complementary region,e.g, a tail. Such a tail region could further be bound to complementarynucleic acid. For example, single stranded nucleic acid can be presentas one or more regions of single stranded DNA as a gap between otherwisecontinuous double stranded structure (see FIG. 3, Gap 2). Alternatively,linear single stranded nucleic acid can be present as tails, or linearsingle stranded nucleic acids in which one end is bound to the CHENACand the other end is free (See FIG. 4 and 6 a). Gaps and tails can besingle stranded RNA or DNA or a variety of other polymers both naturaland synthetic, including modified nucleic acids, nucleic acid analogues,polysaccharides, proteins and other natural and synthetic polymers. Suchsingle stranded regions can serve as a means to segregate biologicalfunction from other functions and as regions of complementarity for thebinding of nucleic acids (as in Example 6b).

[0119] The nucleic acid components can contain one or more nicks inwhich 3′-5′ phosphodiester linkage between constituent bases isdisrupted (See FIG. 1b and 2 b)

[0120] Ligands or chemical modifications can be attached to the nucleicacid, modified nucleic acid or nucleic acid analogue by modification ofthe sugar, base and phosphate moeities of the constituent nucleotides(Engelhardt et al., U.S. Pat. No. 5,260,433, fully incorporated hereinby reference) or to a non nucleic acid segment of the CHENAC such aspolysaccharide, polypeptide and other polymers both natural andsynthetic. Modifications of sugar and phosphate moieties can bepreferred sites for terminal binding of ligands or chemicalmodifications and other moieties. Modifications of the base moieties canbe utilized for both internal or terminal binding of ligands or chemicalmodifications and other moieties. Modifications which are non-disruptivefor biological function such as specific modifications at the 5positions of pyrimidines (Ward et al. U.S. Pat. No. 4,711,955, andrelated divisionals) and the 8 and 7 positions of purines (Engelhardt etal., U.S. Pat. No. 5,241,060 and related divisionals; Stavrianopoulos,U.S. Pat. No. 4,707,440 and related divisionals) may be preferred. Thecontents of each of the aforementioned U.S. patents and their relateddivisionals are incorporated herein by reference.

[0121] Chemical modification can be limited to a specific segment of theconstruct such as a tail or a gap, or dispersed throughout the CHENAC.Thus, the construct may contain at least one terminus, such a terminuscomprising, for example, a polynucleotide tail. Such a modified nucleicacid, subsequently introduced into a cell, could be displaced and/orreplaced.

[0122] In a further embodiment the present invention provides theconstruct, described above, further comprising at least one ligandattached covalently or noncovalently to one or more of the modifiednucleotide analogs, nonnucleic acid entities (or combinations of theforegoing). Such ligands and chemical modifications can be addeddirectly to the CHENAC through covalent and non-covalent interactions.Covalent additions can be made by chemical methods (Engelhardt et al.)and enzymatic incorporation. Non-covalent additions can be made throughnucleic acid-nucleic acid interaction, antigen-antibody interaction,hydrophobic interaction and other interactions based on nucleic acidsequence, nucleic acid structure, protein structure. Indirect additionsto the CHENAC can be made by these same methods and interactions. Whenincluded in the present invention, such ligand or ligands are attachedto any portion or any form of the present construct. Thus the ligand orligands can be attached to a single stranded segment, a double strandedsegment, a single stranded construct tail, a sequence complementary to aconstruct tail or to any combinations of these portions or forms.

[0123] Ligands or chemical modifications, being any chemical entity,natural or synthetic, which can be utilized in this invention includemacromolecules greater than 20,000 m.w. as well as small molecules lessthat 20,000 m.w. The ligand or ligands can include both macromoleculesand small molecules. Macromolecules which can be utilized include avariety of natural and synthetic polymers inlcluding peptides andproteins, nucleic acids, polysaccharides, lipids, synthetic polymersincluding polyanions, polycations, and mixed polymers. Small moleculesinclude oligopeptides, oligonucleotides, monosaccharides,oligosaccharides and synthetic polymers including polyanions,polycations, lipids and mixed polymers. Small molecules includemononucleotides, oligonucleotides, oligopeptides, oligosaccharides,monosaccharides, lipids, sugars, and other natural and syntheticentities.

[0124] Ligands and chemical modifications provide useful properties fornucleic acid transfer such as 1) cell targeting entities, 2) entitieswhich facilitate cellular uptake, 3) entities specifying intracellularlocalization, 4) entities which facilitate incorporation into cellularnucleic acid and 5) entities which impart nuclease resistance.

[0125] 1) Cell targeting entities which can be utilized include:

[0126] a) antibodies to cellular surface components and epitopes

[0127] b) viruses, virus components or fragments of virus componentswhich have affinity for cellular surface components. These include suchproteins as the gp120 protein of HIV which binds to the CD4 receptor ofT4 lymphocytes (Lever 1995 British Medical Bulletin 51;149, incorporatedherein by reference).

[0128] c) ligands which have affinity for cell surfaces. These includehormones, lectins, proteins, oligosaccharides and polysaccharides.Asialoorosomucoid, for example, binds to the cellular asialoglycoproteinreceptor (Wu et al. 1989 J Biol Chem 269;16985, incorporated herein byreference) and transferrin binds to transferrin cellular receptors(Wagner et al. 1992 89; 6099, also incorporated herein by reference).

[0129] d) polycations such as polylysine that bind nonspecifically tocell surfaces (Wu and Wu)

[0130] e) Matrix proteins such as fibronectin that bind to hematopoieticcells and other cells (Ruoslahti et al. 1981 J. Biol. Chem. 256;7277,incorporated by reference),

[0131] f) lectins which bind to cell surface components.

[0132] Entities which facilitate cellular uptake include inactivatedviruses such as adenovirus (Cristiano et al. 1993 Proc Natl Acad Sci USA90;2122: Curiel et al. 1991 Proc Natl Acad Sci USA 88;8850, all of whichare incorporated by reference); virus components such as thehemaglutinating protein of influenza virus and a peptide fragment fromit, the hemagglutinin HA-2 N-terminal fusogenic peptide (Wagner et al.1992 Proc Natl Acal USA 89;7934, also incorporated herein by reference).

[0133] Entities which specify cellular location include:

[0134] a) nuclear proteins such as histones

[0135] b) nucleic acid species such as the snRNAs U1 and U2 whichassociate with cytoplasmic proteins and localize in the nucleus (Zieveand Sautereauj 1990 Biochemistry and Molecular Biology 25;1,incorporated by reference)

[0136] 4) entities which facilitate incorporation into cellular nucleicacid include:

[0137] a) proteins which function in integration of nucleic acid intoDNA. These include integrase site specific recombinases (Argos et al.1986 EMBO Journ 5; 433, incorporated by reference); and

[0138] b) homologous nucleotide sequences to cellular DNA to promotesite specific integration.

[0139] 5) Entities which impart nuclease resistance modifications ofconstituent nucleotides including addition of halogen atoms groups tothe 2′ position of deoxynucleotide sugars. (Braket et al., U.S. PatentApplication Serial No. 07/446,235, filed on Dec. 4, 1989, incorporatedby reference).

[0140] Ligands or chemical modifications can be introduced into CHENACseither a) directly by conjugation, b) by enzymatic incorporation ofmodified nucleoside triphosphates c) by reaction with reactive groupspresent in constituent nucleotides and d) by incorporation of modifiedsegments. These processes include both chemical and enzymatic methods.Enzymatic methods include primer extension, RNA and DNA ligation, randompriming (Kessler et al. 1990 Advances in Mutagenic Research, Vol. 1,Springer Verlag, pp 105-152), nick translation (Rigby et al., 1977, J.Mol. Biol. 113, 237), polymerase chain reaction (Saiki et al., 1985Science 239, 487), RNA labeling methods utilizing T7, T3 and SP6polymerases, (Melton et al. 1984 Nucleic Acids Research 12, 7035; Morriset al. 1986 Gene 41,193), terminal addition by terminal transferase(Roychoudhury et al. 1979 Nucleic Acids Research 6, 1323). Chemicalmethods (described in Kricka, 1995 Nonisotopic Probing, Blotting andSequencing, Academic Press) include direct attachment of ligands orchemical modifications to activated groups in the nucleic acid such asallylamine, bromo, thio and amino; incorporation of chemically modifiednucleotides during chemical synthesis of nucleic acid (Cook et al. 1988Nucleic Acids Research 16, 4077; Stavrianopoulos U.S. Pat. No. 4,707,440and related divisionals), chemical end labeling (Agrawal et al. 1986Nucleic Acids Research 14, 6777); labelling of nucleic acid with enzymes(Jablonski et al., 1986 Nucleic Acids Research 14, 6115). All of theabove-listed publications and U.S. patent are herein incorporated byreference.

[0141] CHENACs can be prepared by the incorporation of nucleic acidsegments modified by ligands or chemical modifications. Constructs canalso be prepared by the incorporation of unmodified nucleic acidsegments together with other segments. Segments incorporated intoconstructs can be single stranded or double stranded or composed of bothsingle and double stranded regions. Such segments can be composed ofDNA, RNA, a combination of DNA and RNA, or chimeric nucleic acids. Allor part of a segment can be; composed of modified nucleic acid ornucleic acid analogue. All or part of a segment can contain natural orsynthetic polymers. A segment can be prepared by any of the chemicalmethods and enzymatic methods listed above.

[0142] The present invention provides for choice of localization ofligands or chemical modifications. In order that such ligands orchemical modifications do not interfere with biological activitysegments with biological activity can be isolated from modified segmentsin the CHENAC. Also, modifications can be confined to a region of asegment. For example, a specific primer labeled with Ligands or chemicalmodifications of choice can be hybridized to a defined region of theconstruct, and polymerization can be done in the presence unmodifiednucleotides in order to confine the ligands or chemical modifications toa defined area of the primer. Alternatively, an unmodified primer can beused to synthesize in the presence of modified nucleotides to confinethe ligands or chemical modifications to the non-primer region of thestrand. Alternatively, by using a primer containing ligands or chemicalmodifications, labeling can done be throughout the strand or throughcomplementarity to a tail.

[0143] Regions of biological activity in constructs can specify codingfor RNA (such as antisense RNA or ribozymes as described in this patent,Example 26) or for RNA which in translated into protein or for DNA.Regions of biological activity in CHENACs can contain sequences forhybridization with intracellular nucleic acid sequences, integrationinto cellular DNA, termination sequences, primer sites, promoter sitesand processing signals and sequences.

[0144] In one preferred embodiment the construct of the presentinvention carries a net positive charge or a net negative charge.Further, the construct can be neutral or even hydrophobic. It should notbe overlooked that the construct may comprise unmodified nucleotides andat least one other member or element selected from one or morenucleotide analogs and non-nucleic acid entities, or both.

[0145] Another significant embodiment of the present invention is aconstruct which when present in a cell produces a product, the constructbeing bound non-ionically to an entity comprising either a chemicalmodification or a ligand addition, or both. As in the case of the otherabove-described construct, this construct may also comprise at least oneterminus, such terminus comprising a polynucleotide tail. Thepolynucleotide tail is hybridizable or hybridized to a complementarypolynucleotide sequence. An antibody to a double stranded nucleic acidcan be directed and thus bound to such hybridized polynucleotide tailsequences. The antibody can comprise a polyclonal antibody or amonoclonal antibody.

[0146] 2. Universal Gene Delivery

[0147] Other useful terms and definitions include the following:

[0148] Nucleic Acid Component: a compound or composition in a cellcapable of producing a product. The composition comprises a nucleic acidsequence desired to be delivered to a cell including polynucleotide,modified nucleic acid and nucleic acid analogues which can be singlestranded or double stranded RNA or DNA, RNA/DNA hybrid molecules andchimeric nucleic acids; nucleic acid construct and chemically modifiednucleic acid constructs (See Examples 1 through 13); viruses includinganimal viruses such as adenovirus, adeno associated virus, retrovirusand plant viruses and bacteriophages; plasmids including the T1 plasmid;or plasmid derivatives that have encapsidated into viral particles byvirtue of packaging signals. Nucleic Acid Components can be produced invivo or assembled in vitro or produced chemically or produced by thetechniques of recombinant DNA. The product produced from the NucleicAcid Component in the cell could be a polynucleotide including mRNA,antisense RNA or DNA, ribozymes or it could be a protein or a proteinproduct.

[0149] Domain: A Domain is an entity that has a segment that binds noncovalently either to a cell or to a Nucleic Acid Component.

[0150] Binder: A Binder is a carrier or matrix that includes at leastone Domain.

[0151] The present invention overcomes the limitations of prior art byproviding a composition and method for universal and efficient nucleicacid transfer. The nucleic acid, whether in a virus vector, in a nucleicacid construct or as polynucleotide, can be introduce into a widevariety of cell types. Furthermore, the use of virus vectors in thisinvention is not limited to a specific or a unique viruses but a widevariety of virus vectors can be used. This invention is universal in tworespects; 1) any Nucleic Acid Component can be applied either in vivo orin vitro and 2) any target cell can be used.

[0152] In the practice of this invention it is possible to:

[0153] 1) bring into close proximity the Nucleic Acid Component and thetarget cell; and

[0154] 2) provide specificity between the Nucleic Acid Component and thetarget cell.

[0155] 3) enhance nucleic acid transfer to the cell by providingCompetence Factors which enhance nucleic acid transfer through enhancingcell growth, cellular uptake of nucleic acid, cellular localization ofnucleic acid and integration of nucleic into cellular DNA.

[0156] The present invention provides materials and methods for thedelivery of nucleic acids to cells. The specificity and/or proximity areprovided through an intermediate, a Binder which consists of at leastone Domain. If the Binder has at least one Domain to the target cell,then the Binder is attached to a Nucleic Acid Component. If the Binderhas at least one Domain to a Nucleic Acid Component, then the Binder isattached to a target cell. If the Binder has at least one Domain to boththe Nucleic Acid Component and the target cell the Domain to the cell isdifferent from the Domain to the Nucleic Acid Component.

[0157] One of the significant embodiments of the present invention is acomposition comprising a non-natural entity which in turn comprises atleast one domain to a nucleic acid component; and at least one domain toa cell of interest. The domain or domains to the nucleic acid componentare different from the domain or domains to said cell. Optional elementsmay be added to this composition or non natural entity including thenucleic acid component, the cells of interest, or both such nucleic acidcomponent and such cells.

[0158] The entity can, of course, comprise a binder. Further, the binderand the domain in the non natural entity can be the same or they can bedifferent

[0159] A Binder is a support or matrix that is composed of at least oneDomain. A Binder can be natural or synthetic, such as a polymer,support, matrix or carrier (or combination of these). The bindercomprises at least one Domain to a Nucleic Acid Component or to a cellof interest or to both. As such, the Binder can be a monofunctional orbifunctional entity. In the case of a monofunctional Binder, only oneDomain is present, either to the Nucleic Acid Component or the cell ofinterest. In the case of a bifunctional binder, at least two domains arepresent, one to the Nucleic Acid Component and the other to the cell ofinterest. Where two domains are present in the binder, i.e., abifunctional binder, the domain to the Nucleic Acid Component isdifferent from the Domain to the cell of interest. In some cases Domainsand Binders can be the same entity, such as an antibody that has asegment (an Fab region) that binds to an epitope and has an Fc segmentthat can function as a support for attachment.

[0160] A Domain is an entity that has a segment that binds either to acell or to a Nucleic Acid Component. Domains can be natural or syntheticpolymers including oligopeptides, polypeptides, oligosaccharides,polysaccharides, oligonucleotides and polynucleotides. These includemonoclonal. antibodies, polygonal antibodies, polycations such aspolyamines, ligands to cell surface proteins, extracellular matrixproteins and ligands and their binding partners. These can be producedin vivo or assembled in vitro or produced chemically or produced byrecombinant DNA techniques.

[0161] Domains provide binding to cells or to NA Entities throughspecific or non-specific binding through a variety of interactionsincluding nucleic acid-nucleic acid interaction, antigen-antibodyinteraction, receptor-ligand interaction, hydrophobic interaction,polyionic interaction and other interactions based on nucleic acidspecificity, nucleic acid sequence and proteins capable of specificallybinding to such sequences or secondary structures or combinationsthereof. Interactions between ligand binding pairs and betweencomplementary nucleic acid sequences may be preferred for theapplication of this invention. These include a nucleotide sequencerecognized by a complementary sequence, an antigen by an antibody, alectin recognized by its cognate sugar, a hormone recognized by itsreceptor, an inhibitor recognized by an enzyme, a cofactor recognized byits cofactor enzyme binding site, a binding ligand recognized by itssubstrate and combinations of the foregoing.

[0162] Antibodies provide useful Domains. Monoclonal and polyclonalantibodies and fragments of these can be used. Antibodies can beobtained from sera, from hybridomas and by recombinant DNA methods.Bispecific antibodies which have the capability to bind to two differentepitopes can also be useful. These can be hybrid hybridomas (Staerz andBevan 1986 Proc Natl Acad Sci USA 83; 1453), heteroantibodies producedby chemical conjugation of antibodies, or fragments of antibodies, ofdifferent specificities (Fanger et al. 1992 Critical Rev Immunol.12;101), bispecific single chain antibodies (Gruber et al. 1994 JournImmunol 152;5368) produced by genetic engineering and diabodies(Holliger et al. 1993 Proc Natl Acad Sci USA 90;6444) produced bygenetic engineering. All of the foregoing publications are incorporatedherein by reference.

[0163] Useful Domains with non-specific cell binding properties includemolecules with polyionic properties such as polycations includingpolylysine, protamine, histones or segments or fragments thereof.

[0164] Useful Domains with specific cell binding properties include:

[0165] 1) those with binding affinity for a natural cell component,epitope or ligand. Such cell binding domains include ligands specific tocell receptors such as hormones, mono- and oligosaccharides, viralproteins which recognize cell receptor sites, extracellular matrixproteins such as fibronectin and fragments thereof, antibodies to cellproteins and fragments thereof.

[0166] 2) those with binding affinity for a non-naturally introducedligand where a) the ligand is attached to a cell by chemical means suchas by reaction with a tyrosine or amino group of a cellular surfaceprotein or b) the ligand is indirectly attached to a cellnon-specifically.

[0167] Useful Domains with non-specific Nucleic Acid Component bindingproperties include those which bind non-covaiently and not through aligand/receptor system. Examples are polyeations such as polylysine andhistones that bind to nucleic acid.

[0168] Useful Domains with specific Nucleic Acid Component bindingproperties include:

[0169] 1) those with binding affinity for a natural component of aNucleic Acid Component, epitope or ligand. These include:

[0170] a) antibodies to nucleic acid including antibodies to doublestranded and single stranded DNA, to double and single stranded RNA orto RNA/DNA hybrids; proteins with nucleic acid binding properties suchas the Cro protein of bacteriophage lambda which binds to a sequence of17 base pairs (Anderson et al. 1981 Nature 290;754, incorporated byreference).

[0171] b) antibodies to an epitope or receptors for a ligand of aNucleic Acid Component. These include antibodies to viral proteins,cellular receptors and virus binding proteins, such as the CD4 proteinof lymphocytes.

[0172] 2) artificial specific binding systems (Domains) can be formed bychemically introducing a ligand to the Nucleic Acid Component where saidligand has a corresponding receptor. Such specific ligands or epitopescan be artificially introduced by chemical modification of a tyrosine oramino group of, for example, a vector virus protein.

[0173] Binders possessing two Domains can exist naturally or they can beprepared synthetically or artificially. For example, a Binder whichpossesses one Domain with cell binding capabilities can be associatedwith a Domain with Nucleic Acid Component binding capabilities to form abifunctional Binder. This association can occur by 1) by covalentattachment 2) by specific non-covalent attachment and 3) by non-specificnon-covalent attachment or 4) as a fusion peptide prepared byrecombinant DNA techniques.

[0174] In the above-described composition of this invention the nucleicacid component can take a number of different forms including a nucleicacid, a nucleic acid construct, a virus, a viral fragment, a viralvector, a viroid, a phage, a plasmid, a plasmid vector, a bacterium, anda bacterial fragement as well as combinations of these. The cell ofinterest can be prokaryotic or eukaryotic. As described elsewhere inthis disclosure the domains can be attached noncovalently or through abinder or through combinations of these. Where noncovalent binding isused, ionic interactions and/or hydrophobic interactions are preferred.In addition the noncovalent binding can comprise a specific complex,e.g., a specific complex mediated by a ligand binding receptor. Theligand binding receptor can itself take a number of forms. Suitable butnot necessarily limited to these members are a polynucleotide sequenceto be recognized by its complementary sequence; an antigen to berecognized by its corresponding monoclonal or polyclonal antibody, anantibody to be recognized by its corresponding antigen; a lectin to berecognized by its corresponding sugar, a hormone to be recognized by itsreceptor; a receptor to be recognized by its hormone; an inhibitor to berecognized by its enzyme; an enzyme to be recognized by its inhibitor; acofactor to be recognized by its cofactor enzyme binding site; acofactor enzyme binding site to be recognized by its cofactor; a bindingligand to be recognized by its substrate; or a combination of theforegoing.

[0175] Another aspect of the present invention concerns the composition,described above, wherein the domain to the nucleic acid component andthe domain to the cell of interest are natural, and the binder isattached to the nucleic acid component by means other than a naturalbinding site. Here, as in other embodiments, the binder can comprisemodified fibronectin or modified polylysine or both.

[0176] Cells of interest containing or associated with theabovedescribed compositions may be contained within a biological system,such as an organism.

[0177] Also provided are methods for introducing a nucleic acidcomponent, as described above, into a cell. Essentially the methodcomprises providing any of the above-described compositions andadministering these to an appropriate biological system. Administrationcan be carried out in vivo or ex vivo.

[0178] This invention also contemplates kits which are useful forintroducing a nucleic acid component into a cell of interest. These kitscomprise in packaged containers or combination a non-natural entitywhich comprises at least one domain to a nucleic acid component, and atleast one domain to the cell of interest. Optionally included in suchkits are the nucleic acid components, the cells of interest and buffersand instructions.

[0179] Another significant embodiment is a composition comprising anentity which comprises at least one domain to a cell of interest,wherein the domain or domains are attached to a nucleic acid componentwhich is in non double stranded form. As elsewhere, the entity cancomprise a binder, and the binder in the domain can be the same or theycan be different. Among others the binder can comprise a polymer, amatrix, a support or a combination of these. The cell of interest can beprokaryotic or eukaryotic. As also described above, the nucleic acidcomponent can take a number of forms including but not limited to anucleic acid, nucleic acid construct, nucleic acid conjugate, a virus, aviral fragment, a viral vector, a viroid, a phage, a plasmid, a plasmidvector, a bacterium, and a bacterial fragment or combinations of these.The domain can comprise covalent bonding or noncovalent binding, orboth. Preferred as noncovalent binding are ionic interactions andhydrophobic interactions (or both), and a specific complex e.g., aspecific complex mediated by a ligand binding receptor. Such ligandbinding receptors have been described above. The cell of interest whichis part of the composition may be contained within an organism. Thislast described composition can likewise be usefully employed in a methodfor introducing a nucleic acid component into a cell. This process hasalso been described above.

[0180] Kits for introducing a nucleic acid component into a cell ofinterest can be fashioned from this composition. Such a kit comprises inpackaged containers or combinations an entity which comprises a domainto a cell of interest, wherein the domain is attached to a nucleic acidcomponent which is in non-double stranded form. Buffers and instructionsmay be optionally included.

[0181] This invention also provides a composition comprising an entitywhich comprises a domain to a nucleic acid component wherein the domainis attached to a cell of interest. As further embodiments of this justdescribed composition are the entity, the binder, the domain, nucleicacid component, the cell of interest, the covalent bonding andnoncovalent binding of the domain, the ionic and hydrophobicinteractions, the specific complex (including its mediation by a ligandbinding receptor), the ligand binding receptor, as well as organisms,methods and kits for introducing nucleic acid components into cellscontaining the cell of interest are all as variously described above.

[0182] Attachment of Nucleic Acid Components to Monofunctional Binders

[0183] 1 Covalent Attachment of a Nucleic Acid Component to aMonofunctional Binder Which Possesses a Domain to a Cell

[0184] Covalent attachment can occur by direct coupling between reactivegroups inherent to a Domain or Binder or by the use of a bifunctionalcrosslinker. Also, reactive groups can be introduced into Domains andBinders in order to facilitate such covalent attachment. Attachment toproteins, for example, can occur through reactive amino groups ortyrosine residues. Attachment can be made by protein-proteinconjugation. Covalent attachment can also be made to polysaccharides andto polynucleotides. Covalent attachment to a nucleic acid, modifiednucleic acid or nucleic acid analogue can be made through modificationof the sugar, base or phosphate moieties of the constituent nucleotides(Engelhardt et al., U.S. Pat. No. 5,260,433, incorporated by reference).Also, nucleotide analogues can be introduced into nucleic acid toprovide reactive groups, e.g., allylamine groups (Ward et al. U.S. Pat.No. 4,7711,955 and divisionals, also incorporated herein by reference)and proteins can be covalently attached to these as described belowusing N-maleimido tri(aminocaproic) acid N-hydroxysuccinimide ester as abifunctional coupler. Modifications of sugar and phosphate moieties canbe preferred sites for terminal attachment of ligands and othermoieties. Modifications of the base moieties can be utilized for bothinternal or terminal attachment of ligands and other moieties.Modifications can include those which are non-disruptive forhybridization such as specific modifications at the 5 positions ofpyrimidines (Ward et al., U.S. Pat. No. 4,711,955 and relateddivisionals). Modifications of the 8 and 7 positions of purines(Englhardt et al. U.S. Pat. No. 5,241,060 and related divisionals) andStavrianopoulos, U.S. Pat. No. 4,707,440 and related divisionals) may bepreferred. In one embodiment, the chemical modification in the constructor construct components may be effected to a moiety independentlyselected from a base, a sugar, and a phosphate, or a combination of anyor all three.

[0185] Direct covalent attachment of a Nucleic Acid Component to aMonofunctional Binder can be illustrated by attachment of a doublestranded DNA molecule (the Nucleic Acid Component) to an antibody whichbinds to a cell surface component (a monofunctional Binder). Forexample, an antibody which binds to the CD4 component of lymphocytes canbe covalently attached to a double stranded DNA (a Nucleic AcidComponent) which has been modified by the incorporation of nucleotidescontaining allylamine in order to provide a primary amine as a reactivegroup. The covalent attachment can be made as described below usingN-maleimido tri(aminocaproic) acid N-hydroxysuccinimide ester as abifunctional coupler.

[0186] Fibronectin can also be used for the covalent attachment of aNucleic Acid Component for delivery of nucleic acid to cells. Forexample, fibronectin, a fibronectin fragment or fibronectin containingcompounds can be attached to either a polynucleotide or to a virusvector. For example, fibronectin can be covalently attached to anallylamine group of a Nucleic Acid Component. A virus vector NucleicAcid Component, such as adenovirus, can also be covalently bound tofibronectin by protein-protein conjugation. The covalent attachment canbe made as described below using N-maleimido tri(aminocaproic) acidN-hydroxysuccinimide ester.

[0187] 2) Specific Non-covalent Attachment of a Nucleic Acid Componentto a Monofunctional Binder Which Possesses a Domain to a Cell

[0188] Non-covalent attachment of a Nucleic Acid Component can occurthrough complementary nucleic acid binding. A Binder composed of anantibody to a cell surface protein can be covalently coupled to a singlestranded DNA by allylamine groups incorporated into the DNA as describedbelow using N-maleimido triaminocaproic acid N-hydroxysuccinimide esteras a bifunctional coupler. The single stranded DNA is attached throughcomplementarity to a tail sequence of a Nucleic Acid Component. Forexample, an antibody to a CD4 cell receptor can be covalently attachedto a single stranded DNA molecule which is complementary to the singlestranded tail of a construct (such as the one described in Example 6) todeliver nucleic acid to CD4 +cells.

[0189] Fibronectin can be modified to provide for the non-covalentattachment of a Nucleic Acid Component. Fibronectin can be covalentlyattached to an antibody which has binding specificity for a virus vectorsuch as adenvirus. Fibronectin and anti-adenovirus antibody arecovalently attached by the use N-maleimido tri(aminocaproic) acidN-hydroxysuccinimide ester as a bifunctional coupler as described below.

[0190] 3) Non-specific Non-covalent Attachment of a Nucleic AcidComponent to a monofunctional Binder Which Possesses a Domain to a Cell

[0191] This can be achieved by the non-covalent attachment of a Domain,such as polylysine which binds to polynucleotides (Nucleic AcidComponent). Polylysine can attach to a monofunctional Binder composed ofa DNA oligomer modified by the covalent addition of trilactyl lysyllysine (Domain to a cell) as described in Example 1 of this patent. Theresulting entity can deliver nucleic acid specifically to liver cells.

[0192] Attachment of Cells to Monofunctionl Binders with Domains to aNucleic ACid Component

[0193] 1) Covalent Attachment of a Cell to a Monofunctional Binder WhichPossesses a Domain to a Nucleic Acid Component

[0194] A Binder with a Domain for a Nucleic Acid Component can becovalently attached to a cell. For example, a monoclonal antibody toadenovirus can be covalently attached to a cell to provide adenovirusbinding sites on the cell surface. Covalent attachment of the antibodycan be made by the use of N-maleimido trifaminocaproic) acidN-hydroxysuccinimide ester as a bifunctional coupler.

[0195] Synthesis of the bifunctional coupler and its use for covalentattachment of proteins is described. Tri(aminocaproic) acid is reactedwith a threefold excess of 3-maleimidopropionic acidN-hydroxysuccinimide ester at a pH 7.8 for 30 minutes at roomtemperature. The pH is adjusted to 4.0 with acetic acid and the solutionis freeze dried. The solid is triturated with ethanol to removeunreacted 3-maleimidopropionic acid active ester and traces of ethanolare removed in vacuum. The solid residue is dissolved indimethyllformamide and filtered from the insoluble salts and reactedwith 1.1 equivalents of dicyclohexyl carbosuccinimide at roomtemperature overnight. The hydroxyurea is removed by filtration and thedimethylformamide is removed in high vacuum at 35° C. The semisolidresidue is triturated with isopropanol to remove unreacteddicyclohexylcarbodiimide and N-hydroxysuccinimide. The solid residue iswashed with absolute ether and the ether traces are removed by vacuumleaving N-maleimido tri(aminocaproic) acid N-hydroxysuccinimide ester(Compound I).

[0196] Cells are treated with ElIman's reagent to block reversibly thiolgroups on the cell surface. The amino groups on the cell surface arereacted with Compound I in isotonic phosphate buffer at pH 7.8 for 30minutes. Excess Component I is removed by centrifugation of the cells at1000×g at room temperature for 5 minutes and decanting the supernatantfluid. The cells are resuspended in phosphate buffered isotonic salineand reacted for one hour at room temperature with an antibody to whichthiol groups have been added. Thiol groups are added to the antibody byreaction with homocysteine thiolactone at pH 9.0.

[0197] At the end of the reaction the cells are reacted with 0.5 mMcysteine in phosphate buffered saline to reconstitute any blocked thiolresidues on the cell surfacae, and the cells are washed bycentrifugation in phosphated buffered saline.

[0198] 2) Specific Non-covalent Attachment of a Cell to a MonofunctionalBinder Which Possesses a Domain to a Nucleic Acid Component

[0199] This can be accomplished by the covalent attachment of biotin tocell surface proteins using an N-hydroxysuccinimide ester of biotin(Enzotin, Enzo Biochem, Inc.). A binder composed of an antibody toadenovirus covalently attached (by the Fc portion) to avidin will bindto biotin molecules on the cell surface to provide adenovirus binding tothe cell surface.

[0200] 3) Non-specific Non-covalent Attachment of a Cell to aMonofunctional Binder with a Domain for a Nucleic Acid Component

[0201] Polylysine can be covalenty attached to the Fc portion of anantibody to adenovirus. The polylysine/anti-adenovirus antibody willbind to the cell surfaces to provide attachment sites for an adenovirusvector.

[0202] Binding of Cells to Nucelic Acid Components through BifunctionalBinder Mediation

[0203] Such bifunctional Binders can be formed by the attachment of twoDomains either directly or through a binder or a matrix. The attachmentcan be covalent, non-covalent, non-specific non-covalent or specificnon-covalent. Specific attachment of cells to Nucleic Acid Componentscan be accomplished by the use of a bifunctional Binder. Such a Bindercan be prepared by the association of a domain for a Nucleic AcidComponent with a Domain for a cell. For example, an antibody toadenovirus can be covalently attached by the Fc portion to polylysine.An antibody to a cell surface protein such as CD4 can also be covalentlyattached to the polylysine to produce a bifunctional Binder.

[0204] A bifunctioal Binder can also be prepared by non-covalent bindingthrough hybridization of complementary nucleic acid strands that havebeen attached to two different antibodies. The Fab fragment of anantibody to adenovirus can be modified by the addition of a homopolymersuch as polythymidilic acid (poly T). The Fab fragment of an antibody toa cell surface marker, such as CD4, also be modified by the addition ofa homopolymer such as, in this case, polyadenylic acid (poly A). The twomodified Fab fragments can be joined by A:T base pairing to provide forthe delivery of adenovirus to CD4 + cells (See Example 16 for theattachment of Fab fragments to homopolymeric polynucleotides.

[0205] In addition to Domains and Binders, other entities can beprovided to enhance nucleic acid transfer. There can be directly orindirectly attached to a Nucleic Acid Component, to a Binder or to aDomain. Attachment can be made by the methods described above for thecovalent and non-covalent attachment of Nucleic Acid Components toBinders and Domains. These entities include;

[0206] 1 ) entities which enhance cell growth. These includeextracellular matrix proteins such as fibronectin, which enhance thegrowth and the transformation efficiency of cells.

[0207] 2) entities which facilitate cellular uptake. These includeinactivated viruses such as adenovirus (Cristiano et al. 1993 Proc NatlAcad Sci USA 90;2122: Curiel et al. 1991 Proc Natl Acad Sci USA 88;8850,all of which are incorporated herein by reference), virus componentssuch as the hemaglutinating protein of influenza virus and a peptidefragment from it, the hemagglutinin HA-2 N-terminal fusogenic peptide(Wagner et al. 1992 Proc Natl Acal USA 89;7934, incorporated byreference).

[0208] 3) entities which facilitate incorporation of nucleic acid intocellular nucleic acid. These include integrase site specificrecombinases (Argos et al. 1986 EMBO Journal 5; 433, also incorporatedby reference).

[0209] 4) entities which function in cellular localization of nucleicacid. These include nuclear proteins such as histones and nucleic acidspecies such as the snRNAs U1 and U2 which associate with cytoplasmicproteins and localize in the nucleus (Zieve and Sautereauj 1990Biochemistry and Molecular Biology 25;1, incorporated by reference).

[0210] Factors unattached to a Nucleic Acid Construct, a Binder or aDomain can also facilitate nucleic acid transfer by increasing thecompetence of cells for nucleic acid transfer. These include factorswhich act to promote cell growth and are be added to target cellsduring, before or after the process of gene transfer in vivo or ex vivo.These include:

[0211] 1) growth factors such as IL-3, IL-6, GM-CSF, Epo and SCF whichstimulate cell growth (Palsson et al., 1993 Biotechnology 11;368: Kolleret al. 1993 Biotechnology 11;358: Koller et al. 1993 Blood 82;378, bothof which are incorporated by reference) and

[0212] 2) entities such as matrix proteins, their fragments or compoundscontaining these moieties, e.g., fibronectin, which form a cell bindingmatrix which promotes cell growth.

[0213] The present invention provides one or more of such effects invivo or ex vivo. Such in vivo or ex vivo effects include the following:

[0214] 1) bringing a Nucleic Acid Component and a target cell into closeproximity

[0215] 2) providing specificity for the interaction between the NucleicAcid Component and the target cell.

[0216] 3) facilitating introduction of the Nucleic Acid Component to thetarget cell.

[0217] 4) enhancing the cells capability to be transformed, i.e., thecompetence of the cell, by providing growth factors, matrix support andother factors.

[0218] 5) providing for localization, integration and stability of theNucleic Acid Component and derivatives of the Nucleic Acid Component inthe cell.

[0219] 6) providing a Nucleic Acid Component or a derivative of it whichin the cell is capable of producing one or more products which includeantisense RNA, antisense DNA, sense RNA, ribozymes, decoys, mRNA andproteins.

[0220] 3. Multimeric Complexes

[0221] The present invention provides novel methods and compositions toform multimeric complexes in which the individual components enjoyretention of their monomeric activity while also maintaining solubilityafter being joined together. Such a multimeric complex consists of morethan one monomeric unit, either bound to each other noncovalentlythrough a polymeric interaction or noncovalently bound to a matrix by apolymeric interaction.

[0222] The present invention provides a multimeric complex compositioncomprising more than one monomeric unit attached to each other throughpolymeric interactions or attached to a binding matrix through polymericinteractions or a combination of both interactions. The polymer oroligomer of the monomeric unit can be linear or branched, and it cancomprise a homopolymer or a heteropolymer. The monomeric unit cancomprise an analyte-specific moiety such as one which is capable ofrecognizing a component in a biological system, e.g., a virus, a phage,a bacterium, a cell or cellular material, a tissue, an organ or anorganism, or combinations thereof.

[0223] The analyte-specific moiety can take a number of forms includingits derivation or selection from a protein, a polysaccharide, a fattyacid or fatty acid ester and a polynucleotide (linear or circular orsingle stranded) or a combination of these. As an analyte-specificmoeity such a protein can comprise an antibody (polyclonal ormonoclonal), a hormone, a growth factor, a lymphokine or a cytokine, anda cellular matrix protein, or a combination of these.

[0224] A monomeric unit is an entity comprised of two elements. Saidfirst element is a compound. Said second element is a polymer (oroligomer) capable of noncovalently binding, complexing or hybridizingeither to the polymer or oligomeric element of a second monomeric unitor to the polymer or oligomer that makes up a binding matrix. Amongothers, the monomeric unit can be selected from a naturally occurringcompound, a modified natural compound, a synthetic compound and arecombinately produced compound or combinations of such compounds.

[0225] Said compound may be an analyte specific moiety that is capableof recognizing and binding to a component in a biological system in vivoor in vitro. A biological system can be comprised of cells, cellularcomponents, viruses, viral components, circulating material,extracellular binding matrices or combinations thereof. The compoundcould be naturally occurring, a modified natural compound, a syntheticcompound or a recombinant product. It could be a polyclonal ormonoclonal antibody, complete protein chains or f(ab) fragments, fromhuman or other species; it could be a lymphokine, cytokine, hormone(e.g., insulin), or growth factor (e.g., erythropoietin) or a cellularmatrix protein (e.g. fibronectin); it could be a ligand, vector,bacterium, or virus; it could be a monosaccharide, oligosaccharide,polysaccharide, polynucleotide, protein, or lipid.

[0226] The polymers can be attached to the compounds either covalentlyor noncovalently. The compounds could be covalently attached to thepolymers through conjugation of reactive groups on the compound and thepolymer. Either the compound or the polymer or both could be chemicallymodified such that conjugation could be facilitated. Either the compoundor the polymer could be modified such that a ligand such as biotin couldbe introduced to one and a receptor for the ligand such as avidinintroduced to the other.

[0227] It is preferred that the polynucleotide segment that is attachedto a given compound does not bind to itself or hybridize together or isnot substantially self-complementary. In the multimeric construct, thecomponent could be homogeneous or heterogeneous, as long as the polymersegment on the homogeneous component or heterogeneous mixture orcompounds can bind or hybridize to the same binding polymer orpolynucleotide in the binding matrix.

[0228] Polymers that are attached to the compounds to form the monomericunits may be selected from the same group of polymers that comprise thebinding matrices with the proviso that they should be able to bindtogether noncovalently.

[0229] The binding matrix is an entity comprised of a linear or branchedpolymeric compound that has more than one portion of a linear segmentthat is capable of noncovalent binding to a linear segment of a polymerof a monomeric unit.

[0230] The linear segment could be comprised of a homopolymer,heteropolymer or co-polymer, a synthetic polymer, a natural polymer, apolynucleotide, modified polynucleotide, or polynucleotide analog orpolyionic compound. Thus the binding matrix can comprise or take itsselection from a polypeptide, a polynucleotide and a polysaccharide orany combination.

[0231] The binding matrix itself may or may not be attached to acompound or an entity. In instances when the binding matrix does attachto a compound or ligand, it is preferred that the binding matrix havereactive groups for such attachment either, directly (covalently) orindirectly (noncovalently) to the compound. The preferred polymers thatare contained within the binding matrix or that are attached to thecompound are those with a monomeric backbone containing a charged group,such as the phosphate backbone of polynucleotides. The hydrogen bondingor ionic state of these polymers could be further changed by thechemical modification of appropriate groups of the side chains orbackbone of such polymers, such as the introduction of chelator goupsdescribed in U.S. Pat. No. 4,843,122 or EP 0 285 057 B1 or amine groupsdescribed in U.S. Pat. No. 4,711,955. All of the contents of theseaforementioned U.S. and foreign patents are incorporated by referenceinto this disclosure.

[0232] The polymer attached to the compound and the polymer of thebinding matrix could bind to each other noncovalently through eitherionic interactions, hydrogen bonding, complementarity or polarinteractions, including dipole-dipole interactions.

[0233] When the binding is through ionic interaction, if the monomericunit contains polycationic segments, then the corresponding bindingmatrix should have polyanionic segments. If the monomeric unit haspolyanionic segments, then the corresponding binding matrix shouldcontain polycationic segments.

[0234] Examples of positively charged polymers could be protamine orpolylysine; soluble DEAE (diethylaminoethyl) cellulose, or DEAE dextran(a branched polysaccharide).

[0235] Examples of negatively charged polymers are techoic acids(polymeric chains of glycerol or ribitol molecules linked to each otherby phosphodiester bridges), polyglutamic acid, carboxymethyl cellulose,dextran sulfate (a branched polysaccharide with 3 negatively chargedsulfate groups), and polyacrylic acid.

[0236] When the binding is through hydrogen bonding or complementarity,if the monomeric unit has a polynucleotide sequence attached, thecorresponding binding matrix should have the complementary nucleic acidsequence.

[0237] Binding matrix polymers preferentially have net ionic charges orsufficient polarity to be soluble and have the capability of noncovalentbinding to another polymer of opposite polarity, charge, orcomplementarity Such a polymer could be single or double strandedpolynucleotide, RNA or DNA, modified or unmodified; polynucleic acidanalogs or any other synthetic polymer that exhibits such properties.

[0238] Double stranded nucleic acid can also act as a polyanionicbinding matrix. In this case the monomeric unit is attached to apolycationic entity such as polylysine or polyamine.

[0239] Another way of constructing such complexes is throughprotein-nucteic acid interactions. Polypeptides that exhibit highaffinity levels for nucleic acids can be attached to desirable compoundsto form monomeric units that can then be complexed together by bindingto a nucleic acid polymer. The sequence of the nucleic acid polymer canbe made up of multimers of binding sequences in the cases where themonomeric units are derived from sequence specific binding proteins suchas the HIV TAR protein. However, the choice of the sequence of thenucleic acid polymer can be completely unrestricted in cases where themonomeric units are derived from sequence independent DNA bindingproteins such as histone.

[0240] One can optimize a given multicomplex compound by adjusting thenumber of monomeric unites in a given binding matrix such that oneobtains the maximum number of compounds on a given binding matrix, whilemaintaining solubility and avoiding stearic hindrance to assure maximalfunctioning of the multicomplex construct.

[0241] When the binding of a monomeric unit to the binding matrix isthrough ionic interaction of two oppositely charged polymers, the ratioof the monomeric unit to the binding matrix has to be adjusted such thatthe net charge or the charge distribution of the multicomplex constructis sufficient to maintain solubility.

[0242] Such multimeric complexes are formed by introducing a polymer toan individual compound that can bind either to another polymer and/orcan bind to a polymer of another compound. In the case ofpolynucleotides, the binding could be through complementary sequences.The polymers could be homopolymers or heteropolymers, sufficient inlength to form a stable bond. In a stable bond formed bypolynucleotides, the polymer could be from approximately 5 to severalthousand nucleotides in length.

[0243] One aspect of these multicomplex units is the formation ofcomplexes with high affinity for the target entity. A multi-antibodycomplex of this invention will exhibit a much higher avidity for thetarget antigen than a single antibody. Such a complex will be usefultherapeutically and for in vitro diagnosis. In vivo such complexes couldbe used as more effective immunologic reagents, including antiviral,antibacterial and antitumor agents. In the case of in vivo use of such amultimeric complex, the preferred polymers are polynucleotides ormodified polynucleotides since nucleic acids are better toleratedimmunologically. For in vitro diagnostics, such multicomplexes could beused to develop more sensitive assay systems. The sensitivity of anydiagnostic system depends on two factors, the sensitivity of the signaland the affinity between the analyte and analyte specific moiety. If theaffinity is not high enough there could be practical or theoreticallimits as to how much one could increase saturated binding in the systemwith the target entity.

[0244] Furthermore, such complexes could be used for efficient genetransformation both in vivo or in vitro (as discussed in thedisclosure).

[0245] A certain concentration of the binding partner is required inorder to obtain a certain level of binding in vivo as well as in vitro,.A multimeric complex of biological binding elements, which upon bindingto a cell can trigger biological effects in the cell, would have a muchhigher binding affinity to a target cell than the correspondingmonomeric unit. Consequently much lower quantities of such amulticomplex compared to the monomeric unit would be needed to achievethe same physiological effect. Examples of such biological complexes arehormones, cytokines, lymphokines, growth factors, ligands. Amulticomplex of insulin could be useful in that manner in diabetictreatment

[0246] In addition to being used to form more potent biologicaleffectors, multimeric complexes or polymeric units of this invention canbe used to form multimeric complexes or polymeric units of compoundswhich bind to etiological agents, such as viruses, bacteria and fungi,or to toxic compounds. These binding compounds could be polyclonal ormonoclonal antibodies, complete protein chains or F(ab) fragments, fromhuman or other species; or the receptor protein of the etiological agentor toxic compound. The binding of such polymers or complexes to thetarget is stronger than the binding of the monomers and these polymersor complexes can recognize and bind to low concentrations of theetiological agent or the toxic compound. These compositions can beapplied, therefore, for more effective therapeutic use against infectionand toxicity. These products can be adminstered to patients in vivo orcould be used ex vivo for neutralization of potentially infected ortoxic blood.

[0247] In preparing such complexes, one would modify a compound, suchthat the binding of the compound does not interfere with its biologicalfunction or effects. The preferred attachment of reactive groups oroligomers or polymers covalently or through a complex would be vianon-disruptive chemistry. Binding is through reactive groups in thecompound that are not within the active site, binding site or functionalgroups and binding is such as to allow maximal freedom with the leastamount of disruption to the molecule.

[0248] If desired, the spacing of the monomeric units can bepredetermined by defining the nature of the region that the monomericunits are bound to the matrix to optimize their spacing so as to provideproper co-operative binding and also to reduce potential stearichindrance. An example of this type of disposition of the monomeric unitsis shown in FIG. 23 from Example 18 where each monomeric unit has beenjoined to a specific unique sequence that is complimentary to differentportions of the M13 binding matrix.

[0249] These multicomplex compounds could further contain many otherentities as ligands, receptors, chemical modifications that eitherenhance their biological function, increase their solubility, providefurther cooperative overall binding or provide capability to bind todesired cells in vitro and in vivo. Thus another aspect of thisinvention is the composition, described above, further comprising anentity attached to the binding matrix. Such an, entity can comprise aligand or a compound which increases the binding of the binding matrix.Examples of such entities are the cellular matrix proteins(fibronectin), lectins, polysaccharides, and polycationic polymers suchas polylysine and histones.

[0250] Any of the above-described compositions can be formulated ashomogeneous forms or compositions or heterogeneous forms orcompositions.

[0251] The above-described multimeric complex composition (and itsvarious embodiments) can be usefully employed in a process fordelivering a cell effector to a cell. In such a process one wouldprovide the multimeric complex composition wherein the monomeric unit ofthe composition comprises a cell effector and administer the compositioneither in vivo or ex vivo. In addition the multimeric complexcomposition can be employed irn a process for delivering a gene or agene fragment to a cell. Here, one would provide the multimeric complexcomposition wherein the monomeric unit comprises the gene or genefragment to be delivered and would administer such composition either invivo or ex vivo as the case may be.

[0252] Another useful multimeric composition comprises more than onecomponent attached to a charged polymer. The charged polymer is selectedfrom a polycationic polymer, a polyionic polymer, a polynucleotide, amodified polynucleotide and a polynucleotide analog as well ascombinations of the foregoing. Such a component can comprise a protein,e.g., an antibody (polyclonal or monoclonal), an F(ab′)₂ fragment orboth. The antibody can be further complexed with a target comprising anenzyme.

[0253] 4. Intron Inactivation

[0254] The present invention provides (1) a universal composition forconditional nucleic acid processing by the introduction of a processingelement into a nucleic acid sequence produced from a constructintroduced into a cell. Said produced nucleic acid is processed in acompatible cell, i.e., a cell capable of processing RNA by removal ofthe processing element. Said RNA is not processed in an incompatiblecell, i.e., a cell capable of processing RNA by removal of theprocessing element and (2) a binary biological function in which asingle nucleic acid construct bearing at least two operons ortranscriptional units non-native to a cell when introduced into saidcell results in the protein gene product of one of the operons impactingthe the protein gene product(s).

[0255] The present invention provides a novel method and constructs forcapability for the conditional inactivation of a gene by the use of anon-native, or heterologous, processing element which only permits geneexpression in compatible cells. The method utilizes the introduction ofa heterologous processing element into the coding region of a desiredgene resulting in inactivation of the gene when present in anon-compatible cell. The intron can be inserted at a number sites inmost genes. The heterologous processing element carries no flankingsequences, and thus introduces no additional sequences upon insertion.In a preferred embodiment, the gene product either is absent or inactivein an incompatible cell, but when introduced into a compatible cellyields a functional mRNA molecule which, upon translation, the geneyields an unaltered protein.

[0256] Among the significant embodiments is a nucleic acid constructwhich when introduced into a cell expresses a non native polymerase, thepolymerase being capable of producing more than one copy of a nucleicacid sequence from the construct. This construct can further comprise arecognition site for the non native polymerase. Such a recognition sitecan be complementary to a primer for the non native polymerase. Theprimer preferably comprises transfer RNA (tRNA).

[0257] In certain embodiments the non native polymerase comprises amember selected from DNA polymerase, RNA polymerase and reversetranscriptase as well as any combination of the foregoing enzymes. TheRNA polymerase preferably comprises a bacteriophage RNA polymerase,e.g., T3, T7, and SP6, or combinations thereof. Furthermore, theabove-described construct can comprise a promoter for the RNApolymerase.

[0258] The nucleic acid produced from the construct can take a number offorms including but not limited to DNA, RNA, a DNA-RNA hybrid and aDNA-RNA chimera, or combinations thereof. The DNA or RNA can comprisesense or antisense, or both.

[0259] Another significant aspect of this invention concerns a nucleicacid construct which when introduced into a cell produces a nucleic acidproduct comprising a non native processing element which when in acompatible cell, the processing element is substantially removed duringprocessing. The processing element can comprise an RNA processingelement including but not limited to an intron, a polyadenylation signaland a capping element, or combinations of the foregoing.

[0260] The nucleic acid product can be single stranded and it cancomprise any of antisense RNA, antisense DNA, sense RNA, sense DNA, aribozyme and a protein binding nucleic acid sequence, as well ascombinations of any of these. The protein binding nucleic acid sequencepreferably comprises a decoy that binds a protein required for viralassembly or viral replication.

[0261] Also provided by this invention is a process for selectivelyexpressing a nucleic acid product in a cell, the product being such thatfurther processing is required for its functioning. The processcomprises as its first step providing a nucleic acid construct whichwhen introduced into a cell produces a nucleic acid product comprising anon-native processing element, which when in a compatible cell, theprocessing element being substantially removed during processing. Thesecond step comprises introducing this construct into the cell. Theprocessing element, e.g., an RNA processing element, the nucleic acidproduct and the steps of introducing the construct in vivo and ex vivoare all as described previously. Significantly, in this process, theconstruct can be introduced into a biological system containing thecell. This biological system can comprise, an organism, an organ, atissue and a culture (cell or tissue) as well as combinations of these.

[0262] The present invention provides a universal method for utilizingprocessing elements, including heterologous elements, for conditionalgene inactivation. Rather than a restriction enzyme site, the frequentlyoccurring sequence (C/A)AGG post splice junction sequence is used as theinsertion site. This site results from the consensus sequence resultingfrom an excision of an intron. The consensus splice sequence for splicedonors is (CIA)AG*GU and the consensus sequence for splice acceptors is(U/C)_(n)N(C/U)AG*G where * represents the splice site (Mount 1982 Nucl.Acids Res. 10, 459). The frequent occurence of this sequence providesnumerous potential sites for the insertion of processing elements.Insertion at any of these sites in a gene coding region should notaffect subsequent removal of the processing element in a compatiblecell. Proteins produced from processed mRNA should demonstrate no changein amino sequence or enzyme activity since only processing elementsequences free of flanking exon sequences are introduced therebyallowing the processing event to regenerate the original codingsequence.

[0263] Furthermore, the site of insertion for a processing element doesnot appear to affect gene expression. Mayeda and Oshima (1990 Nucl.Acids Res. 18: 4671, incorporated by reference) showed that a nativeintron, isolated as a restriction fragment of DNA containing theβ-globin intron with the conserved bases of the 3′ end of the donor exonattached, could be introduced into various sites of a cDNA copy ofβ-globin and subsequently be spliced out normally, irrespective ofintron location in the β-globin coding sequence. This is consistent withthe consensus sequences that have been identified for splice donors andsplice acceptors and that there are no particular requirements for aspecific sequence at the 5′ end of the acceptor axon.

[0264] It is possible that insertion of a heterologous processingelement may not in all cases inactivate a gene when present in anincompatible cell. Although splicing has been observed in procaryoticsystems for bacteriophage T4 (Chu et al. 1984 Proc. Nat. Acad. Sci. USA81: 3049, incorporated by reference), it is in this case due to aself-splicing intron (Chu et al., 1985 J. Biol. Chem. 260: 10680,incorporated by reference) and thus independent of processes employed incompatible cells. Therefore, in a procaryotic environment, the intronshould remain in the mRNA as long as a self-splicing intron is not used.In addition, if the number of bases in the intron is a multiple of 3,the reading frame remains the same and a fusion protein with additionalamino acids derived from the intron sequence could potentially beproduced. These extra bases may or may not change the activity of thetarget protein depending upon the nature of the extra amino acids andthe insertion site within the protein coding sequence. A preferred modeof inactivation is the use a heterologous processing element thatintroduces a frame shift mutation and/or a stop codon(s).

[0265] The present invention also provides for the introduction of genesnot native to a cell into said cell wherein the protein products of suchintroduced gene(s) interact with and impact other proteins produced fromintroduced non-native gene(s).

[0266] The non-native protein gene products resulting from an introducednon-native gene(s) can impact another non-native protein by a variety ofprocessess including polymerization; activation; facilitating transport;competitive inhibition; allosteric interaction; chemical modificationincluding phosphorylation, dephosphorylation, methylation,demethylation, proteolysis, nuclease activity, glycosylation; andothers.

[0267] Non-native genes can be introduced into cells as RNA, DNA or bothDNA and RNA. Non-native genes can be introduced into a cell linkedtogether on a single nucleic acid construct or introduced separately ondistinct constructs. Introduction of non-native genes into cells can bedone by any of a variety of methods for gene delivery (reference).

[0268] The present invention provides the following benefits:

[0269] a) This invention has utility for the conditonal inactivation ofgenes when such genes would be lethal to the host cell or when suchgenes present in a host cell introduce a danger. Thus, genes which wouldbe impossible to clone, such as those which code for enzymes whichdestroy bacterial cell walls, can be inactivated by intron insertion andthus cloned in this form in a bacterium. Genes coding for toxicproducts, including tetanus toxin, risin, pseudomonas toxin, E. colienterotoxins, cholera toxin and other plant, animal and microbialtoxins, can be inactived and maintained stably and safely in anincompatible cell and activated to produce an unaltered gene product ina compatible cell. This has special application to cell killing genetherapy.

[0270] b) The present invention provides utility for the inactivation,in incompatible cells, of the expression of polymerase catalysts whoseexpression can be realized in compatible cells. This has application toexpression of a variety of gene products, either RNA or protein, undercontrol of promoters of a variety of polymerases. Polymerases, nativeand non-native to the cell, that could be used in this way include RNApolymerases from T3, T7 and SP6.

[0271] c) This invention provides for normally incompatible genes to becloned together on the same nucleic acid construct. For example, asingle construct can be designed containing sequences for the productionof T7 promoter directed transcript(s) of choice and T7 RNA polymerase.The ability to clone such genes on the same nucleic acid constructrather than as separate constructs provides the following benefits:

[0272] i) The efficiency of cotransfection of the two genes is 100%.

[0273] ii) In the case of T7 RNA polymerase and a nucleic acid sequencefor T7-directed transcript of choice, the entire functional unit issufficiently compact that it can be cloned into a vector which can onlyaccept inserts below a certain size limit as, for example, adenoassociated virus which can only accept inserts of 4.7 kilobases or belowand remain functional (Muzyczka 1992 in Current Topics in Microbiologyand Immunology, Springer Veriag, Heidelberg, 158;97, incorporated byreference)

[0274] d) Another application of this invention provides for theinteraction of non-native gene or its protein products in a cell wherethe interaction of the introduced genes and/or their gene products canyield useful intracellular processes for gene therapy applications.

[0275] In an application of the present invention, an intron isintroduced into the coding sequence of T7 RNA polymerase in a constructthat also contains a T7 promoter directing the transcription of a usefulgene product. As discussed earlier, the use of T7 polymerase forsynthesis of a gene under control of a T7 promoter has been accomplishedin-compatible cells, but always in the context of placing the twoentities on separate constructs, i.e., the T7 RNA polymerase and thegene under the control of a T7 promoter are used as a two-part system.The present invention (see Examples) describes the conditionalinactivation of a gene (that normally does not a contain a processingelement) by the precise introduction of an intron between the last twoG's of a site that has the post splice junction sequence (C/A)AGG. Theintroduction of an intron into sites with this sequence creates afunctional splice donor and a functional splice acceptor. Therefore, aconstruct with this modification should lack any expression of T7 RNApolymerase in an E. coil cell, but the normal coding sequence can berestored from transcripts after introduction into a compatible cell.This allows the construction of a single construct that contains boththe T7 RNA polymerase and, for example, antisense directed by a T7promoter, with lethality to an incompatible cell being avoided byintroducion of the heterologous processing element into the polymerasecoding sequence. In a compatible cell, normal expression of thepolymerase will occur but lethality should be negated by the nature ofits environment. First, the autocatalytic cascade, due to transcriptionaround the circular plasmid, believed to be responsible for lethality ofE. coli, would not occur in stably transformed mamalian cells formed byintegration into the chromosomal DNA. Second, in the presence ofconcatameric integration of the construct, runoff transcription from theT7 promoter past a T7 terminator sequence into the coding sequence forthe polymerase should produce RNA that would be translated with very lowefficiency due to the lack of appropriate signals for processing,transport and translation.

[0276] The same advantages of this invention that are enjoyed for theproduction of T7 directed RNA, such as antisense RNA, can be applied tothe T7 RNA polymerase directed production of protein.

[0277] 5. Hairpin Construct

[0278] The introduction of genetic material into cells can be done bytwo methods. One method is the exogenous application of nucleic acidswhich act directly on cellular processes but which themselves are unableto replicate or produce any nucleic acid. The intracellularconcentrations of these molecules that must be achieved in order toaffect cellular processes is dependent on the exogenous supply. Anothermethod for nucleic acid delivery is the introduction into cells ofPrimary Nucleic Acid Constructs which themselves do not act on cellularprocesses but which produce single stranded nucleic acid in the cellwhich acts on cellular processes. In this case the introduced PrimaryNucleic Acid Construct can integrate into cellular nucleic acid or itcan exist in an extrachromosomal state, and it can propagate copies ofitself in either the integrated or the extrachromosomal state. Thenucleic acid consstruct can produce, from promoter sequences in thePrimary Nucleic Acid Construct, single stranded nucleic acids whichaffect cellular processes of gene expression and gene replication. Suchnucleic acids include antisense nucleic acids, sense nucleic acids andtranscripts that can be translated into protein. The intracellularconcentrantions of such nucleic acids are limited to promoter-dependentsynthesis.

[0279] Definitions:

[0280] Primary Nucleic Acid Construct. A composition consisting ofnucleic acid which in a cell propagates Production Centers.

[0281] Production Center. A nucleic acid molecule derived from a PrimaryNucleic Acid Construct which in a cell is able to propagate otherProduction Centers or to produce single stranded nucleic acid. As usedherein, the term production center is intended to cover secondarynucleic acid components which can be produced from a primary nucleicacid construct. Also covered are a tertiary nucleic acid component whichcould be produced from the secondary nucleic acid component, as well asany nucleic acid product which may be produced from the secondarynucleic acid component.

[0282] Propagation. The generation or formation of a Production Centerfrom a Primary Nucleic Acid Construct or the generation or formation ofa Production Center from another Production Center. However, productioncenters cannot produce a Primary Nucleic Acid Construct.

[0283] Production. The generation of a single stranded nucleic moleculesfrom a Production Center.

[0284] Inherent Cellular Systems. Cellular processes and componentspresent in cells which can be utilized for the Production andPropagation as well as the function of single stranded Nucleic AcidProducts. Such processes and components can be native to the cell, or beintroduced to the cell by artificial means or by infection by, forexample, a virus.

[0285] The effectiveness of single stranded nucleic acids produced fromPrimary Nucleic Acid Constructs is dependent on their concentration, thestability and the duration of production in the cell. Current methodsfor achieving intracellular concentrations are limited by a dependenceon promoter directed synthesis.

[0286] The present invention provides a novel composition construct andmethod whereby single stranded nucleic acid is produced in the cell fromtemplates which are formed in the cell and derived from Primary NucleicAcid Constructs in said cell. This invention further provides for aPrimary Nucleic Acid Construct which, when introduced into a cellPropagates one or more Production Centers each of which in the cell iscapable of Production of single stranded nucleic acid product.

[0287] One aspect of the present invention provides a means to attainhigh intracellular levels of single stranded nucleic acid throughamplification. Such amplification occurs by the Propagation from aPrimary Nucleic Acid Construct of more than one Production Center andfrom each Production Center one or more single stranded nucleic acids.However, Production Centers are not capable of producing Primary NucleicAcid Constructs.

[0288] Thus, a significant embodiment of this invention concerns acomposition comprising a primary nucleic acid component which uponintroduction into a cell produces a secondary nucleic acid componentwhich is capable of producing a nucleic acid product, or a tertiarynucleic acid component, or both. The secondary and tertiary nucleic acidcomponents and the nucleic acid product are incapable of producing theprimary nucleic acid component. In this composition the cell can ofcourse be eukaryotic or prokaryotic.

[0289] In the present composition, the primary nucleic acid componentcan comprise a nucleic acid, a nucleic acid construct, a nucleic acidconjugate, a virus, a viral fragement, a viral vector, a viroid, aphage, a phage vector, a plasmid, a plasmid vector, a bacterium, and abacterial fragment or combinations of any of these.

[0290] Primary Nucleic Acid Constructs consist of single or doublestranded nucleic acid (or even partially double stranded) or composed ofboth single and double stranded nucleic acid, and the nucleic acid canbe RNA, DNA or a combination of RNA and DNA. The nucleic acid can beunmodified or it can be modified to provide desirable properties. Forexample, modified bases can be incorporated to provide nucleaseresistance, interaction with Inherent Cellular Systems, cellularlocalization and other properties for nucleic acid constructs asdescribed in this disclosure. Furthermore, the primary nucleic acidcomponent can comprise nucleic acid analogs which likewise can be usedin combination with DNA, RNA, or both.

[0291] Primary Nucleic Acid Constructs can reside in the cell integratedinto chromosomal DNA or as extrachromosomal entities. The PrimaryNucleic Acid Construct, as an integral part of a chromosome, can bereplicated concomitant with chromosomal DNA during cell divisionprocesses or it can be replicated as part of an extrachrosomal elementcontaining DNA replication elements, such as sequences for origin ofreplication and others.

[0292] Primary Nucleic Acid Constructs contain sequence information forthe Propagation of Production Centers and for the subsequent Productionof single stranded product, Thus, for this purpose, a variety ofdesirable elements can be encoded in a Primary Nucleic Acid Construct.Production Centers and Primary Nucleic Acid Constructs may contain oneor all of these elements. These include regulatory elements such aspromoters and enhancers; primer binding sites; processing elements suchas intron sequences, poly A sequences, sequences specifying capping andtermination sequences; sequences specifying cellular localization signalsequences with affinity for cellular proteins. Primary Nucleic AcidConstructs can also contain sequences for the synthesis of proteinswhich act to propagate Production Centers. For example, sequences for anucleic acid polymerase which acts to propagate a Production Center canbe present in a Primary Nucleic Acid Construct (See Example 20 of thispatent).

[0293] Primary Nucleic Acid Constructs can propagate Production Centersthrough the activity of nucleic acid polymerizing catalysts present asInherent Cellular Systems. Production Centers can be RNA, DNA or acombination of RNA and DNA. They can be single stranded, double strandedor contain both single and double stranded regions. Production Centerscan propagate other Production Centers and/or produce single strandednucleic acid product with biological activity directly or through theactivity of Inherent Cellular Systems.

[0294] Production Centers can produce a variety of single strandednucleic acids such as antisense RNA sequences, antisense DNA sequences,ribozyme sequences and mRNAs which can be translated into proteins canall be produced. Desirable properties to enhance biological activity canalso be incorporated. Thus, RNA processing signals, sequences specifyingcellular location, sequences for binding cellular proteins and otherfunctions can be incorporated into single stranded nucleic acidsproducts.

[0295] As production centers, the secondary nucleic acid component andthe tertiary nucleic acid component (as well as other subsequentcomponents, e.g., a quaternary nucleic acid component, can comprise DNA,RNA, a DNA-RNA hybrid, and a DNA-RNA chimera or a combination of theforegoing.

[0296] When the above-described compositions further comprise a signalprocessing sequence, such sequences can be selected from a promoter, aninitiator, a terminator, an intron, and a cellular localization elementor a combination of these. Such signal processing sequences can becontained in any of the elements of the composition including thoseselected from the primary nucleic acid component, the secondary nucleicacid component, the nucleic acid product and the tertiary nucleic acidor a combination of these. The nucleic acid product can of course besingle stranded as well as comprising antisense RNA, antisense DNA, aribozyme and a protein binding nucleic acid sequence or combinations ofthese. Preferred as a protein binding nucleic acid sequence is a decoythat binds a protein required for viral assembly or viral replication.

[0297] In these above-described compositions, production of anycomponent or nucleic acid product can be mediated by a vector, preferredvectors comprise viral vectors, phage vectors, plasmid vectors, as wellas combinations of these.

[0298] The present composition can be incorporated into a cell which iseukaryotic or prokaryotic. The composition can be introduced either invivo or ex vivo into such a cell.

[0299] Also contemplated by the present invention are production centersincluding the secondary or tertiary nucleic acid components or thenucleic acid product which can be produced from the composition.

[0300] The Propagation of Production Centers from Primary Nucleic AcidConstructs, the Propagation of Production Centers from other ProductionCenters and the Production of single stranded nucleic acid fromProduction Centers can proceed by a variety of processes which derivefrom sequences present in these structures (as described above) and fromInherent Cellular Systems. Inherent Cellular Systems involved in theseprocesses include RNA polymerases, RNA processing enzymes, DNApolymerases, Reverse Transcriptases, Ribonuclease H, endonucleases,exonucleases including ribozymes, enzymes involved in nucleic acidrepair, nucleic acid ligases, cellular nucleic acids acting as primers,and entities involved in nucleic acid replication, transcription,translation, localization of nucleic acid in the cell, transport ofnucleic acid, integration of nucleic acid into cellular nucleic acid andothers.

[0301] Elements for Propagation and Production include: 1) single ormultiple promoters, 2) self priming processes, 3) one or more primerbinding sites., and 4) multiple priming.

[0302] 1) Promoters for Propagation and Production can be present in oneor more copies in a Production Center or in a Primary Nucleic AcidConstruct. Such promoter sequences can be present in a preexisting andfunctional form, as, for example, in a double stranded DNA PrimaryNucleic Acid Construct introduced into a cell. Functional promotersequences can also form subsequent to introduction of a Primary NucleicAcid Construct into a cell. For example, a single stranded RNA PrimaryNucleic Acid Construct containing promoter sequenceses which arenon-functional (since they are present as single stranded ribonucleicacid) can be converted to functional promoter sequences by propagationin the cell to a double stranded DNA Production Center from said PrimaryNucleic Acid Construct. This Propagation can be achieved by the presencein the Primary Nucleic Acid Construct of primer binding sites, such asthe HIV primer binding site for which lysyl tRNA acts as a primer, andreverse transcriptase as an Inherent Cellular Element. The generation ofdouble stranded DNA in this way forms a functional promoter.

[0303] Functional promoter sequences can also be generated by theformation of double stranded regions from self complementary formationin a single stranded Primary Nucleic Acid Construct. For example, thepresence of both the sense and antisense sequences for a promoter and acoding sequence under its control can be present in a single strandedDNA Nucleic Acid Product or Production Center. Self hybridization ofthese regions of the same molecule can generate a functional promoter inthe formed double stranded region of this single stranded molecule.

[0304] 2) A single stranded Primary Nucleic Acid Construct can Propagateor a linear single stranded Production Center can Propagate or Producenucleic acids by a self priming process. In this process, the 3′ end ofsuch a molecule can hybridize with complementary regions locatedelsewhere in the molecule and act as a primer for the synthesis ofcomplementary nucleic acid. For example, the 3′ end of a linear singlestranded RNA can act as a primer for a polymerase such as reversetranscriptase.

[0305] 3) One or more primer site sequences can be included in a PrimaryNucleic Acid Construct or in a Product Center. Sequences for the primerbinding site of a retrovirus, such as HIV, which utilizes lysyl tRNA asa primer, can be included in one or more copies in a single stranded RNAPrimary Nucleic Acid Construct or Production Center. Lysyl tRNA issupplied as an inherent cellular system. In the presence of reversetranscriptase, Propagation and Production of complementary DNA proceedsfrom the primer site.

[0306] 4) Multiple priming processes can be utilized for Production andPropagation. For example, a double stranded Primary Nucleic AcidConstruct composed of one DNA strand and one RNA strand can be actedupon by nucleases to generate limited endonucleolytic cleavage in theRNA strand. The resulting fragments can act as primers the productionand propagation of DNA synthesis as catalyzed by inherent cellularprocesses such as reverse transcriptase.

[0307] 6. U1 Antisense System

[0308] This invention provides a composition of matter comprising anucleic acid component which when present in a cell produces anon-natural nucleic acid product, the product comprising two elements: aportion of a localizing entity and a nucleic acid of sequence. Theportion of the localizing entity is preferably sufficient to permitlocalization of the non natural nucleic acid product. Furthermore, theportion of the localizing entity preferably comprises a cytoplasmic ornuclear localization signaling sequence.

[0309] The nucleic acid sequence of interest can comprise various formsof nucleic acid including but not limited to DNA, RNA, a DNA-RNA hybridand a DNA-RNA chimera or combinations of these. When comprising RNA, thenucleic acid of sequence preferably comprises a nuclear localized RNAwhich may be complexed with protein molecules. Among such nuclearlocalized RNA are the so called snRNAs. Preferred as snRNA's are U1, orU2, or both.

[0310] The non natural nucleic acid product can be of course singlestranded and it may comprise varibous members or forms including thoseselected from antisense RNA, antisense DNA, sense RNA, sense DNA, aribozyme, and a protein binding nucleic acid sequence. As describedelsewhere, such a protein binding nucleic acid sequence preferablycomprises a decoy that binds a protein involved or required for viralassembly or replication. In another aspect of the present composition,the non natural nucleic acid product comprises antisense RNA orantisense DNA and the portion of the localizing entity comprises anuclear localization signalling sequence. In yet another aspect of thecomposition, the non-natural nucleic acid product comprises antisenseRNA or antisense DNA and the portion of the localizing entity comprisesa cytoplasmic localization signalling sequence. Still yet another aspectconcerns the composition wherein the non-natural nucleic acid productcomprises sense RNA or sense DNA and the portion of a localizing entitycomprises a cytoplasmic localization signalling sequence.

[0311] As described elsewhere the nucleic acid component can takevarious forms, e.g., a nucleic acid, a nucleic acid construct, a nucleicacid conjugate, a virus, or fragment, a viroid, a phage, a plasmid, avector, a bacterium, or fragment, as well as any combination of these.Such nucleic acid can comprise DNA, RNA, a DNA-RNA hybrid and a DNA-RNAchimera and combinations thereof. The nucleic acid can be modified; thecell can be eukaryotic or prokaryotic. The production of nucleic acidproduct is mediated by a vector such as a viral vector, a phage vector,or a plasmid vector or such combinations.

[0312] As described elsewhere the present combination can beincorporated or delivered into a cell which can be eukaryotic orprokaryotic. Introduction into the cell can be ex vivo or in vivo. Thepresent invention also contemplates biological systems (an organism, anorgan, a tissue, a culture) containing the cell into which thecomposition has been introduced.

[0313] The present invention further contemplates a process forlocalizing a nucleic acid product in a eukaryotic cell. In this process,the above-described composition of matter would be provided andappropriately introduced into a eukaryotic cell or a biological systemcontaining such cell. The characteristics of the localizing entityportion, the nucleic acid product, methods, ex vivo and in vivointroduction in this process are all as described above.

[0314] The present invention describes a method and composition forutilising snRNAs as carriers for antisense RNA while retaining theadvantageous features of snRNA for nuclear localization. The presentinvention utilizes removal of sequences from snRNA and their replacementwith desirable sequences such as antisense or sense sequences.

[0315] The correct choice of the site for replacement of a portion ofthe snRNA sequence should not alter the stability and nuclearreimportation features. Digestion of a clone of the human U1 operon withBcI I and Bsp E II (FIG. 41) eliminates a sequence of 49 bases involvedin the formation of the A and B loops formed by U1 RNA (FIG. 41).Removal of this sequence thus both makes room for the addition offoreign sequence and eliminates binding of some snRNP proteins tpusenabling the foreign sequence to be available for antisense inhibitionfree of potential steric hindrance by bound proteins. Elimination of theA and B loops should still allow formation of the C and D loops whichare important for maintaining the re-importation signal (FIG. 41). Thecontinued presence of this secondary structure at the 3′end as well asbinding of splicesome proteins should also have the effect ofmaintaining the stability of the RNA.

[0316] This invention should be applicable to other species of snRNAincluding U2.

[0317] U1 constructs prepared as described for this invention can bedelivered to cells as all or part of nucleic acid constructs by any ofseveral methods applicable to gene delivery.

[0318] 7. Multi-Cassette Constructs

[0319] The present invention, which has application to gene therapy, isa Nucleic Acid Entity which, when introduced into a cell, directs thesynthesis of more than one specific entity from a separate functionalunit, or cassette. The synthesis of each product entity is initiatedfrom its own initiator signal in a cassette. Multi-targeting can beachieved by inclusion of independent cassettes in a singleMulti-Cassette Construct. The advantages of a Multi-Cassette are:

[0320] a) Each entity is formed independently from other entities andthe total number of procuct entities will be a summation of the productsgenerated in the cell by each initiation site.

[0321] b) Interaction with a target by an independently generatedproduct entity should have no effect upon the activity of otherindependently generated product entities.

[0322] c) An integration event that disrupts expression from onecassette should have no effect upon other cassettes in the construct.

[0323] d) Each product entity present in a construct can be directed toa different intracellular locus by use of appropriate signals for eithernuclear or cytoplasmic localization. In situations where productentities acting in the nucleus are combined in the same construct withentities acting in the cytoplasm, the application of Multi-CassetteConstructs allows independent synthesis of the two entities, therebyallowing each to accumulate at its most effective site of action.

[0324] This invention provides a nucleic acid component which uponintroduction into a cell is capable of producing more than one specificnucleic acid sequence. Each such specific sequence so produced aresubstantially nonhomologous with each other and are either complementarywith a specific portion of a single-stranded nucleic acid of interest ina cell or are capable of binding to a specific protein of interest in acell.

[0325] In this component, the single stranded nucleic acids of interestcan be part of the same polynucleotide sequence or part of differentpolynucleotide sequences. The single stranded nucleic acids of interestcan comprise viral sequences. The present nucleic acid component can bederived or selected from any of nucleic acids, nucleic acid constructs,nucleic acid conjugates, a virus or fragment, a phage, a plasmid, abacterium, or fragment, a vector (viral, phage, plasmid), as well as anycombinations of these. The nucleic acid can comprise DNA, RNA, andnucleic acid analogs (or combinations thereof). The DNA and RNA can bemodified.

[0326] In addition, the nucleic acid component can comprise either morethan one promoter or more than one initiator, or both. Furthermore, thespecific nucleic acid sequence products can be produced independentlyfrom either different promoters, different initiators, or combinationsof both. Still further, the specific nucleic acid sequence products canbe either complementary to a viral or cellular RNA or bind to a viral orcellular protein or a combination of such things. The complementaryspecific nucleic acid sequence products can be capable of acting asantisense. The viral or cellular protein can comprise a localizingprotein or a decoy protein which are described elsewhere. Suchlocalizing proteins preferably comprise a nuclear localizing protein ora cytoplasmic localizing protein. Specific nucleic acid sequenceproducts can comprise antisense RNA, antisense DNA, a ribozyme, and aprotein binding nucleic acid sequence or a combination of the foregoing.

[0327] The nucleic acid component can further comprise a means fordelivering the component to a cell containing the nucleic acid ofinterest or the specific; protein of interest. Such delivering means areknown in the art as well as described elsewhere in the disclosure.

[0328] The Multi-Cassette Constructs can be prepared as RNA or DNA. Thenucleic acid can be delivered to the cells as modified or unmodifiednucleic acid or as modified or unmodified RNA or DNA complexed toproteins, lipids or other molecules or as modified or unmodified RNA orDNA as components of pseudo virions, bacteriophage or other viraldelivery systems.

[0329] Multi-Cassette constructs can be delivered to target cells bymethods commonly used for gene transfer as described in thisapplication.

[0330] The presence of independent synthesis units, i.e., cassettes, ina Multi-Cassette Construct provides versatility for the presentation ofproduct entities to the cell through the choice of product entities,synthesis initiator signals and other elements. A Multi-CassetteConstruct can be designed to code for a variety of product entities.Thus, cassettes can be designed to code for synthesis of RNA, DNA orprotein and such cassettes can be assembled in various combinations in asingle Multi-Cassette Construct.

[0331] Elements can be incorporated into each cassette to regulate theindependently and differentially, if desirable the synthesis, characterand nature and activity of the product entity in the cell. Such elementsinclude the type of promoter, enhancer sequences, RNA processingelements such as introns, cellular localization elements such as nuclearor cytoplasmic localizaton signals and poly A addition signals toprovide for addition of poly-A to mRNA.

[0332] Useful product entities produced by each cassette includeantisense RNA, sense RNA, ribozymes antisense DNA, nucleic acidsequences which bind protein molecules such as decoys which bindproteins required for virus replication: enzymes; toxin molecules;proteins which act in cellular localization of RNA and proteinmolecules; DNA polymerases; reverse transcriptases; RNA polymerases andnucleic acid sequences under control of cognate promoters for such RNApolymerases; proteins which impart viral resistance to a cell (such asinterferons); antibodies and/or fragments thereof; proteins which arrestcell division ; proteins which localize in the cell membrane includingcellular receptors for viruses, hormones, growth factors and otheragents which interact at the cell surface;

[0333] Intracellar synthesis of product entities can be controlled bythe choice of promoter or initiating element. Thus, a cassette can bedesigned which contains sequences for a product entity whose synthesisis under control of an inducible promoter providing for temporalsynthesis of product entities. This provides advantages to applicationswherein, for example, constant production of the product entity wouldhave deleterious effects for the host cell or organism, but whose shortterm effects are beneficial. For example, induction of a product entitywhich arrests cell division processes can impart to the cell virusresistance where virus replication is dependent on such cellularprocesses. In order to restore the cellular processes at a later time,induction can be terminated. Induction can be mediated by use ofpromoters which can be induced by small molecules such as antibiotics,hormones and heavy metals such as zinc. Alternatively, in cases whereconstant production of a product entity or entities is beneficial, apromoter not subject to induction can be utilized.

[0334] Promoters can also be chosen on the basis of their efficiency. Incases where high levels of product entities are required promoters whichinitiate transcription at a high frequency can be utilized.Alternatively, when lower levels of product entities are desirable lessefficient promoters can be used.

[0335] Independently synthesized product entities produced from the sameMulti-Cassette Construct can act at the same target site. For example,in order to increase effectiveness, a series of antisense RNA productentities directed at a viral nucleic acid target site which demonstratessequence variability, such as one of the highly variable regions of thenucleic acid of HIV, can be designed to include the predominantlyoccuring sequences encountered in the wild type HIV population.

[0336] Independently synthesized product entities produced from the sameMulti-Cassette Construct can also act at separate target sites. Forexample, an RNA antisense transcript can be directed at mRNA coding fora particular gene product and a different antisense transcript can bedirected against an m RNA coding for another gene product.

[0337] 8. Virus Resistance

[0338] The present invention involves the use of agents that in vivo actto increase resistance to viruses by gene therapy by interfering withvirus-cell interaction and thus enhancing antiviral gene therapy in-thecell. The interaction of regions on viruses with specific sites on thecell surface, i.e., virus-cell interaction, and the susceptibility ofextracellular virus to immunological agents provide the basis forsupplemental treatment. Agents that act by these means to decrease theeffective levels of virus would provide benefit for gene therapytreatments utilizing antisense.

[0339] As a supplement to gene therapy, the above agents can beadministered to the patient either prior to, concurrently or after agene therapy procedure by intramuscular, intravenous, intraperitoneal,by inhalation or other appropriate means.

[0340] Examples of agents that can interfere with the interaction of avirus and a target cell include:

[0341] a) agents such as antibodies to viral epitopes and cellularproteins which bind viruses. An example of the latter are cellularreceptors recognized by viruses, as, for instance the CD4 receptor thatis recognized by HIV.

[0342] b) agents that stimulate the production of entities that complexwith viruses. These include adjuvants that enhance immunologicalresponses which can be used as a general stimulant and viral antigensthat can be used to induce a specific response;

[0343] c) agents that bind to a target cell and compete with orotherwise slow the entry of a virus into a cell. Viral proteins, such asthe gp124 protein for HIV, that are involved in cell binding could beused in this way. Antibodies to viral proteins can also act in this way.

[0344] In the practice of this invention, additional enhancement can beachieved by the further administration of small molecules such asprotease inhibitors or nucleoside analogues. The additional treatmentcan be either applied prior to, after or concurrently with applicationof the present invention. The current invention has application to thetreatment of virus infections and infections by other intracellularpathogens.

[0345] Thus, the present invention provides a process for increasingcellular resistance to a virus of interest. The process comprises twosteps. First are provided transformed cells phenotypically resistant tothe virus: and a reagent capable of binding to the virus or to avirus-specific site on the cells. Second, the reagent is administered toa biological system containing the cells to increase the resistance ofthe cells to the virus of interest.

[0346] The biological system can comprise an organism, an organ, and atissue or combinations thereof, viral resistant cells can be eukaryoticor prokaryotic. Such cells can further comprise a nucleic acid sequenceselected from antisense RNA, antisense DNA, sense RNA, sense DNA, aribozyme, and a protein binding nucleic acid sequence or combinationsthereof.

[0347] The virus binding reagent can take various forms including butnot limited to an antibody, a virus binding protein, a cell receptorprotein and an agent capable of stimulating the production of a virusbinding protein or combinations thereof. The antibody can comprise ofcourse a polyclonal or monoclonal antibody which can be specific to anepitope of the virus of interest. The virus binding protein preferablycomprises a CD4 receptor; the cell receptor protein preferably comprisesa gp24 protein. In addition the production stimulating agent isselectable from an immunological response enhancing adjuvant and a viralantigen or both.

[0348] The reagent can be administered in vivo or ex vivo to the cells.Moreover, the process of the instant invention can further compriseadministering an additional viral resistance enhancing agent, e.g., aprotease inhibitor, a nucleoside analog, or both.

[0349] In carrying out the present process the additional viralresistance enhancing agent can be administered before, after, or atabout the same time that the binding reagent is administered.

[0350] Also contemplated by this invention are biological systems withincreased viral resistance, such resistance having been obtained by anyof the processes described above.

[0351] 9. Dislocation

[0352] The present invention is a novel method of altering theconcentration of cellular products in a cellular location by theintroduction of a construct that produces a product, the dislocatonagent, which acts to transport cellular entities from one cellularlocale to another. The dislocation agent contains a specificity oraffinity domain by which the dislocation agent binds the cellularentity. Dislocation of the cellular entity is mediated by the bounddislocation agent. The resulting co-localization transports the cellularentity to a cellular location that is different from its functionallocation.

[0353] In contrast to previous work (Izant and Sardelli, 1988, Cottenand Birnstiel, 1989, the contents of both publications incorporatedherein by reference), which sought to localize the genetic products oftheir constructs to a cellular location favorable for antisenseactivity, the present invention acts to disrupt a viral or cellularprocess by dislocation of macromolecules involved in the viral orcellular processes. Thus, due to the presence of an affinity domain onthe dislocation agent, a target molecule will be bound and thentransported to a cellular location determined by the dislocation agent.

[0354] The application of this invention is through the introductioninto cells of nucleic acid constructs which contain sequences for theexpression of RNA. The RNA, acting as the dislocation agent, can itselfcontain sequences for an affinity domain and can transport cellularnucleic acid molecules or proteins to cellular localizations where theyare not normally present. Alternatively, the RNA can bind a targent RNAmolecule and chaperone it to another cellular location where it can'tfunction by the binding of a protein which transports the RNAdislocation agent and its hybridized target RNA to an unnatural cellularlocation. Also the RNA can contain a sequence that when translatedyields a protein dislocation agent with an affinity domain.

[0355] In the current invention, active steps are taken upon theinteraction of the target with the dislocation agent. Examples of wherethis might be useful are RNA molecules that contain signals specifyingtransport from the cytoplasm into the nucleus. Binding of such an RNAdislocation agent to a cytoplasmic RNA or protein would lead toco-localisation of the target into the nucleus. These transportedentities would be unable to function due to their presence in anunnatural cellular location. In a similar way, a protein dislocationagent with an affinity domain for a particular RNA sequence or foranother protein can be designed such that it also has a nuclearlocalisation signal present in its sequence. In this way a targetentity, normally present in the cytoplasm, would be localised in thenucleus.

[0356] This invention provides a nucleic acid construct which whenintroduced into a cell produces a non-natural product. The non naturalnucleic acid product comprises two components: a binding componentcapable of binding to a cellular component; and a localization componentcapable of dislocating the cellular component when bound to the product.The product from this construct can comprise a protein or a nucleic acidor both. The protein can comprise an antibody, e.g., a polyclonal ormonoclonal antibody, such as one directed to a cellular component insidethe cell. Such cellular components can comprise any of the followingincluding but not limited to a nucleic acid, a protein, a virus, aphage, a product from another construct, a metabolite and an allosteariccompound, or combinations thereof. When comprising a protein thecellular component can comprise a viral or non-viral enzyme, a genesuppressor, a phosphorylated protein, e.g., an oncogene, or combinationsthereof.

[0357] The binding component of the product produced from the presentconstruct is selectable from a nucleic acid, a protein and a bindingentity or combinations thereof. The nucleic acid can comprise a sequenceselected from a complementary sequence to the cellular component and asequence to a nucleic acid binding protein or combinations of both. Theprotein is selectable from an antibody, a receptor and a nucleic acidbinding protein or combinations thereof. The binding entity is capableof binding metabolites. The localization component is selectable from anuclear localizing entity, a cytoplasmic localizing entity, and a cellmembrane localizing entity or a combination thereof. The localizingcomponent in the present construct can comprise a member selected from anucleic acid sequence, a nucleic acid structure, e.g., a stem and loopstructure and a peptide or oligopeptide, or combinations of theforegoing.

[0358] The present invention further provides a process for dislocatinga cellular component in a cell. In this process there are provided anucleic acid construct which when introduced into a cell produces anon-natural product, which product comprises two components. First,there is a binding component capable of binding to a cellular component;and second, a localization component capable of dislocating the cellularcomponent when bound to the product. The nucleic acid construct isintroduced into a cell of interest or a biological system containing thecell or cells of interest.

[0359] The following is a list or summary of candidate pairs offered forillustration if not by way of limitation. Potential pairs of relocationagents and their targets are presented.

[0360] An application of the present invention for the dislocation ofcellular macromolecules is the use of a nucleic acid construct thatcontains a nucleic sequence for a U1 snRNA molecule in which a portionof the U1 sequence has been substituted with a sequence unique to aportion of the HIV genome (described previously). In this case the U1RNA in association with snRNP proteins acts as the dislocation agent andthe HIV anti-sense sequence represents the affinity domain. The returnof U1 to the nucleus, as part of normal cellular processing of U1, whilehybridized to target HIV mRNA dislocates the HIV RNA and makes itunavailable for translation in the cytoplasm.

[0361] Another application of this invention utilizing U1 RNA is thesubstitution of HIV packaging signal sequences for a portion of the U1sequence. Introduction of the substituted U1 as part of a nucleic acidconstruct used to transfect cells, provides for the synthesis of adislocation agent containing the U1 RNA sequences and the HIV packagingsequence signals as the affinity signal. The dislocation agent in thiscase binds to essential HIV proteins responsible for forming virions andtransports them from the cytoplasm to the nucleus, thereby inhibitingthe packaging of viral RNA.

[0362] Another application of present invention is the use of a nucleicconstruct which produces an RNA molecule which contains sequencesspecific for splice junctions of HIV RNA as the affinity domain andsequences for the Rev Responsive Element (RRE) of HIV as an affinitydomain for binding to HIV Rev protein molecules which acts as thedislocation agent. In HIV-infected cells, the Rev protein dislocationagent binds to RRE sequences on the RNA which is in turn bound to thesplice junction of the HIV RNA. The complex would be transported by theRev protein to the cytoplasm where the unspliced HIV mRNA would benon-functional.

[0363] Another application of the present invention is the use of RNAsignals for the dislocation of proteins essential for virus replication.The HIV Rev protein is found principally in the nucleolus. However, inthe presence of RNA containing RRE sequences, the Rev protein is foundprincipally in the cytoplasm. Therefore, the presence of a nucleic acidconstruct containing sequences for the cellular producton of an RNAdislocation agent containing RRE sequences would actively remove the Revprotein from the nucleus and induce its relocation in the cytoplasmwhere it would be unavailable for transport of viral RNA. Here the RREsequences in the transcripts act as the affinity domain.

[0364] The many examples which follow are set forth to illustratevarious aspects of the present invention, but are not intended to limitin any way the scope of the invention as more particularly set forth inthe claims below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES Example 1 Preparationof a Two Segment CHENAC in Which the Ligands and Chemical Modificationsare Localized in One Region of One Segment

[0365] (i) Description of Construct

[0366] A construct is prepared from one unmodified strand segment and amodified primer segment (FIG. 1 a). The unmodified single-strandedcircle is derived from a plasmid that contains the desired sequences forbiological function and it also contains an F1 packaging signal.(Plasmids of this nature are available from a variety of commercialsources.). An E. coli host containing this plasmid is infected with M13helper phage to obtain single-stranded DNA packaged into phageparticles. DNA can then be prepared by a variety of commonly usedprocedures. The oligomer primer is synthesized with an allylaminephosphoramidite (prepared by the method of Cook et al., 1988) and thenmodified with tri-lactyl lysyl lysine as described below. The unmodifiedsegment contains a sequence complementary to the modified primersegment. After exposure of the construct to the target cells, thegalactose moieties provide binding to their natural receptor andtransport the complex into the cell. In the present example, the primeris extended by DNA polymerases in the cell to convert the construct todouble-stranded form. (FIG. 1b) which allows the construct to expresssequences specifying biological function in the unmodified region of theCHENAC (designated by the solid black region in FIG. 1b). In thisexample the biological function region of the construct is separatedfrom the region bearing the ligands and chemical modifications.

[0367] (ii) Preparation of Lactyl Isothiocyanate

[0368] p-Nitrophenyl-β-D lactopyranoside (Toronto Research Chemicals,Inc. Catalog # N50385) is converted into p-lsothiocyano-β-Dlactopyranoside by the method described by Rafestin et al. (FEBS Letters40 62-66, 1974). 1

[0369] (iii) Preparation of Trilactyl Derivative

[0370] 0.7 g LysylLysine dihydrochloride (Sigma Chemicals) is dissolvedin 30 ml of H₂O. 4 g of p-lsothicyano-β-D lactopyranoside (approximately8 mMoles) from step (i) is added and the reaction is stirred for 4 hoursat room temperature. During this time the pH of the mixture was adjustedto 9.0 and maintained at that value by the addition of 0.2M NaOH. At theend of the reaction, the volume is adjusted to 500 ml with H₂O andloaded onto a DEAE-DE52 cellulose column (previously adjusted to pH 9.0and then equilibrated with 0.05 M TRIS buffer, pH 9.0). UnreactedLysylLysine remained unabsorbed to the column and is removed by washingthe column with 0.01 M LiCl. The product is eluted with 0.1 M LiCl andthe fractions from the column are analyzed for UV absorbance at 260 nm.The peak is collected and the H₂O evaporated under vacuum. The dryresidue is triturated with an ethanol/ether (3:1) mixture to remove theLiCl, leaving a solid product. The yield of tri-Lactyl-LysylLysine isapproximately 80%.

[0371] (iv) Activation of tri-Lactyl-LysylLysine

[0372] 0.5 g of tri-Lactyl-LysylLysine (0.25 mMoles), prepared in step(iii), is dissolved in 30 ml of dry Dimethylformamide. 1 g ofN-Hydroxy-succinimide is added, followed by 50 mg ofDicyclohexylcarbodiimide. The reaction is allowed to proceed overnightat room temperature. The following day it is evaporated under vacuum.The residue is triturated two times with 25 ml of isopropanol for 30minutes each at room temperature to remove unreactedDicyclohexylcarbodiimide and the excess of N-Hydroxysuccinimide. Theproduct is then washed over a filter with absolute ether, the etherremoved and the product used without any further purification.

[0373] (v) Lactosylation of the Nucleic Acid Portion

[0374] 1 mg of an oligomer designed to be the primer shown in FIG. 1 isdissolved in 4 ml of 0.7M LiCl, 0.1 M bicarbonate buffer (pH 7.8). 20 mgof tri-Lactyl-LsylLysine active ester (an approximately 10-fold excessof the reagent compared to the number of allylamine groups) prepared instep (iii) is dissolved in 1 ml of Dimethylformamide and added and themixture was stirred for 6 hours at room temperature. The mixture isevaporated under vacuum and subsequently dissolved in 1 ml of H₂O. Thesolution is centrifuged to remove insoluble material and the supernatantwas subjected to G50 column chromatography and the DNA fractionscombined.

Example 2 A Double-stranded Version of Example 1

[0375] The construct described in FIG. 1a from EXAMPLE 1 is again usedbut prior to exposing the DNA to the target cells, the primer isextended in vitro by the action of Klenow enzyme (Klenow fragment of DNApolymerase I) to convert the construct into the completelydouble-stranded DNA molecule shown in FIG. 1b. Primer extension isperformed under appropriate conditions to avoid strand displacement, forexample by carrying out the synthesis at 14° C. so that the newlysynthesized strand stops at the position of the 5′ end of the primer.

Example 3 Preparation of a Two Segment CHENAC in Which One Segment hasDispersed Ligands and Chemical Modifications

[0376] (i) Description of the Construct

[0377] A construct is prepared from an unmodified strand segment and amodified primer segment (FIG. 2). The modified segment is a DNA oligomerprepared by chemical synthesis such that it contains allylaminedeoxyuridine bases as described previously. Peptides are synthesizedthat contain sequences for a) a fusogenic peptide derived from influenza(Lear and DeGrado, 1987, J. Biol. Chem. 262: 6500 ) and b) a peptidepromoting localisation to the nucleus of a cell (Kalderone et al., 1984,Cell 33: 499). The peptides are joined to the allyl amine moieties bythe procedure given below. The modified primer is complementary to aregion in the unmodified segment. The primer is hybridized to theunmodified segment and extended by Klenow enzyme in the presence of anucleoside triphosphate mixture containing lactyl-deoxyuridinetriphosphate precursors (described below) using the sequence of theunmodified segment as template. Synthesis (polymerization) of thenascent strand is performed at 14° C., so that extension stops at theposition of the 5′ end of the primer (FIG. 2b).

[0378] (ii) Synthesis of Peptides for Addition into the DNA Primer

[0379] The sequence coding for the Fusogenic Peptide(Gly-Phe-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Leu-Glu-Gly-Gly-Trp-Glu-Gly-Met-Ile-Ala-Gly)and the sequence coding for the Nuclear Localisation Peptide aresynthesized chemically with an additional cysteine group added onto thecarboxy terminus of each.

[0380] (iii) Addition of Peptides to Allylamines

[0381] The allylamine modified nucleic acids are reacted with a 10-foldexcess of 3-maleimidopropionic acid N-Hydroxy succinimide ester in 0.7 MLiCl, bicarbonate buffer (pH 7.9) and incubated at room temperature for40 minutes. At the end of the reaction, the pH is adjusted to 6.0 withacetic acid. The unreacted NHS ester (and its hydrolysis product) areremoved by extraction with n-butanol two times. The DNA is precipitatedwith 4 volumes of Ethanol at −70° C. The pellet is then resuspended in0.1M sodium acetate buffer (pH 6.0) in a minimum concentration of 1mg/ml. The derivatized DNA is mixed with the desired amount ofthiol-containing fusogenic and nuclear localisation peptides from step(ii) and reacted at room temperature for 6 hours. The unreactedmaleimido residues on the DNA are quenched by the addition ofβ-mercapto-ethanol.

[0382] iv) Synthesis of Lactyldeoxy UTP

[0383] 10 μmoles allylamino deoxyUTP (Enzo Biochem, Inc.) are dissolvedin 6 ml of 0.7M Lithium Chloride, 0.2M sodium bicarbonate, pH 7.8 andmixed with 20 μmoles of the lactyl-isothicyanate (described previously)dissolved in 2 ml of Dimethylformamide. The mixture was reacted for 40minutes at 25° C. and then diluted to 100 ml with distilled water andloaded onto a 100 ml bed volume DEAE Sephadex A25 column. The column waswashed with 100 ml 0.05 M triethylammonium bicarbonate buffer (pH 7.8)and the product was eluted with a linear gradient of 0.05 M -0.6 Mtriethylammonium bicarbonate buffer (pH 7.8). The fractions with maximalUV absorbance at 290 nm were collected and the triethylammoniumbicarbonate was removed in vacuo in the rotary evaporator at 35° C. Thesolid residue containing the lactyideoxy UTP is dissolved in 10 mM trisbuffer pH 8.0 and used as a substrate for DNA polymerase.

Example 4 Preparation of a Two Segment CHENAC in Which One Segment hasDispersed Ligands and Chemical Modifications Incoporated byRibonucleotide Moieties

[0384] A single-stranded DNA construct is derived as described inExample 1. A second strand made up of RNA is made by incubation of theDNA template with RNA polymerase and a mixture of ribonucleotidesaccording to the method described in Stavrianopoulos et al. (1972, Proc.Nat. Acad. Sci. 69; 2609). Two types of modified ribonucleotides areincluded in this mixture; lactyl-UTP and allylamine UTP. The allylamineUTP is commercially available (ENZO Biochem, Inc.) and the lactyl-UTP issynthesized as previously described for the lactyl-deoxy-UTP in Example1 except the ribo derivative of allylamine UTP is used as the startingmaterial. After the RNA strand is synthesized, it is separated from theDNA template strand by melting and then the the allyamine nucleotideswere modified further by the addition of fusogenic peptides as describedpreviously in Example 3. The strands were then allowed to reanneal toform the final structure shown in FIG. 3.

Example 5 Preparation of a Three Segment CHENAC Containing a ModifiedSingle Stranded Tail

[0385] (i) Description of the Construct

[0386] This construct is prepared from two unmodified complementary DNAsegments (Segments 1 and 2) and a modified DNA segment (Segment 3).Segment 1 and Segment 2 are hybridized together to form a gapped circlewith the gapped region being complementary to Segment 3. The finalassembly of these segments are shown in FIG. 4. The methods for creatingthe individual components and assembling them into the final constructare given below

[0387] (ii) Preparation of the Gapped Circle

[0388] a) Segment 1 is prepared from plasmid DNA as described previouslyin Example 1. However, in this particular example, the starting plasmidcontains the F(+) packaging signal. Since single-stranded DNA is not asuitable substrate for most restriction enzymes, a small portion of thecircular single-stranded DNA is transformed into double-stranded form byhybridization with an oligo that is complementary to an appropriaterestriction site. In this example, the restriction enzyme is Sma I andthe oligo has been modified by the inclusion of biotinylated nucleotides(Cook, et al. 1988) at the ends. After digestion, the Sma I digestedduplex DNA is destabilized and the biotinylated oligo has a much loweraffinity. Purification of the cleaved single-stranded linear DNA isachieved by passing the digest over a strepavidin column and collectingthe material that does not bind.

[0389] b) Segment 2 is prepared by preparation of two complementaryolgonucleotides (GAP-1 and GAP-2) and hybridizing them together to forman unmodified double stranded oligonucleotide whose sequence willconstitute the gap in the construct. The starting plasmid is the sameone that was used to make Segment 1, except it contains the F(−)packaging signal. The introduced oligonucleotide (GAP-1/GAP-2) containsterminal restriction sites for the restriction enzyme Sma I in order tofacilitate its insertion by restriction digestion and ligation. Aftercloning of a plasmid with the oligonucleotide inserted into the propersite, circular single-stranded Segment 2 DNA is obtained as shown inFIG. 5.

[0390] c) Segments 1 and 2 are annealed together to form a gapped circlewhere the single-stranded region contains the GAP-2 sequence. Theoverall process of steps ii-a, ii-b and ii-c are shown in FIG. 5

[0391] (iii) Synthesis of Segment 3

[0392] Segment 3 is prepared by synthesizing an oligomer similar toGAP-1 which differs from this oligomer in not having the Sma I sitesadded onto the end and also by being synthesized with allylaminemoieties. After synthesis of the oligomer, the allylamine-modifiednucleotides are further modified by the addition of the trilactyl lysyllysine derivative as described previously. Segment 3 was processedfurther by the steps given below.

[0393] (iv) Addition of Modified 3′tail to Segment 3

[0394] 1 mg of the lactosylated oligomer (Segment 3) is dissolved in 10ml of a reaction mixture containing 0.2 M cacodylate (pH 6-8), 1 mMdeoxythymidine Triphosphate, 0.3 mM allylamine-deoxyuridinetriphosphate, 1 mM cobalt chloride, 1 mM β-mercaptoethanol and 40,000units of terminal transferase. The mixture is incubated for 2 hours at35° C. and stopped by the addition of EDTA. Enzyme is removed byabsorption to a phosphocellulose column at pH 6.0 and the flow-throughis collected, precipitated with ethanol and redissolved in 2 ml of 0.1mM EDTA. The final product has a poly-dT tail with approximately ¼ ofthe bases containing allylamine groups. Fusogenic peptides are thenadded onto the allylamine moieties as described previously.

[0395] (v) Final Assembly

[0396] The final construct shown in FIG. 4 was formed by thehybridization of the gapped circle created in step (ii-c) with thetailed oligomer created in step (iv) through the complementary of theGAP-1 and GAP-2 sequences.

Example 6 Preparation of a Three Segment CHENAC Containing an UnmodifiedSingle Stranded Tail Capable of Hybridizing to Homopolymers ContainingLigands

[0397] This construct was created in the same manner as the constructdescribed in Example 5, except that after synthesis of the oligomer forSegment 3, the fusogenic peptide was added to the allylamine derivativesinstead of the lactyl derivatives and the synthesis of the 3′ tail wascarried out in the presence of unmodified dATP. As in the previousexample, Segment 1, Segment and Segment 3 were assembled together tomake a double stranded circle with a 3′ single-stranded tail. However,as shown in FIG. 6 a further step was added in which segment 4 was addedto the complex. This segment was formed by extension of a Thyminetetranucleotide with Terminal transferase in the presence of a mixtureof TTP and the lactyl-dUTP in a ratio of 3:1 using the same conditionsdescribed previously. Hybridization of Segment 4 to the complex resultsin the final construct shown in FIG. 6.

Example 7 Construction of an RNA Derived CHENAC

[0398] A construct is made with the appropriate structure shown in FIG.7. Transcription is carried out in vitro by use of a T7 promoterdirecting the synthesis of the sequences of interest. The transcriptcontains a) sequence A B, which represents a sequence complementary to alactylated DNA primer (prepared as described previously), b) sequence CD which represents a CMV promoter for directing synthesis of atranscript in vivo, c) sequence E F which represents a sequence forbiological function which will be expressed after transcription by theCMV promoter and d) sequence G H which is designed such that itscomplementary sequence will be a primer binding site similar to the oneused by HIV to bind a cellular tRNA^(lys) as a primer for reversetranscriptase. After transcription of the RNA in vitro, the modifiedprimer is annealed to the RNA to form the complex shown in FIG. 7. Thiscomplex could be used either in vivo, ex vivo or in vitro to bind theRNA to a target cell through a ligand/receptor interaction. Afterendocytosis, some portion of the the RNA should be available in thecytoplasm for further processing and activity. FIG. 8 shows the pathwaythat would occur in the presence of reverse transcriptase activity. Thisactivity can be provided either by targeting a cell that has thisactivity already present (either intrinsically or due to a retroviralinfection) or by introducing it by any of a variety of means known tothose skilled in the art. The end result of the steps shown in FIG. 8 isa double stranded linear piece of DNA which will be capable of producingtranscripts that provide a desirable biological activity.

Example 8 Construction of an RNA Derived CHENAC with Multiple Primers

[0399] A construct is made with the appropriate structure shown in FIG.9. Transcription is carried out in vitro by use of a T7 promoterdirecting the synthesis of the sequences of interest. The construct inthis example is similar to the one described in Example 8 except that itis intended to produce an RNA that will be annealed with multipleprimers rather than a single modified primer. One or more of theseprimers can be modified. In the present example, the transcript containsa) sequence A B, which represents a sequence complementary to alactylated DNA primer (prepared as described previously), b) sequence CD, which represents a sequence complementary to a modified DNA primerthat has fusogenic peptides attached (prepared as described previously)c) Sequence E F, which is an unmodified primer d) sequence G H whichrepresents a CMV promoter for directing synthesis of a transcript invivo, e) sequence I J K which represents a sequence for biologicalfunction which will be expressed after transcription by the CMV promoterand d) sequence L M which is designed such that its complementarysequence will be a primer Binding site similar to the one used by HIV tobind a cellular tRNAIYS as a primer for Reverse Transcriptase. For thepurposes of clarity, the appended modifications are not depicted in FIG.10. After transcription of the RNA in vitro, the primers described aboveare annealed to the RNA to form the complex shown in FIG. 9. Thiscomplex could be used either in vivo, ex vivo or in vitro to bind theRNA to a target cell through a ligand/receptor interaction. The ligandmodified primer will promote uptake of the complex and after endocytosisthe fusogenic peptide modified primer will promote the release of theRNA from the endosomes. FIG. 10 shows the pathway that would occur inthe presence of Reverse Transcriptase activity. This activity can beprovided either by targeting a cell that has this activity alreadypresent (either intrinsically or due to a retroviral infection) or byintroducing it by any of a variety of means known to those skilled inthe art. The end result of the steps shown in FIG. 10 is a series ofdouble stranded linear piece of DNA (each initiated from one of theprimers from the complex formed in vitro) which will be capable ofproducing transcripts that provide a desirable biological activity.

Example 9 Construction of a One-Segment Single-Stranded CHENAC

[0400] A construct is made with the appropriate structure shown in FIG.11. Transcription is carried out in vitro by use of a T7 promoterdirecting the synthesis of the sequences of interest. The transcriptcontains a) sequence J K, which represents a sequence complementary to alactyl lysyl lysine modified DNA primer (prepared as describedpreviously) as well as sequences for biological function which include aCMV promoter for directing synthesis of a transcript, a sequence forbiological function which will be expressed after transcription by theCMV promoter and a sequence or sequences complimentary to tRNA bindingsites. This example differs from the two previous examples in that thecomplementary DNA is synthesized in vitro by using Reverse Transcriptasewith the tri lactyl-LysylLysine modified DNA segment as a primer. Theresulting RNA/DNA double stranded molecule is treated with Rnase H toyield a single stranded DNA CHENAC.

[0401] This complex could be used either in vivo, ex vivo or in vitro tobind the DNA CHENAC to a target cell through a ligand/receptorinteraction. After endocytosis, some portion of the the DNA should beavailable in the cytoplasm for further processing and activity. FIG. 12shows two possible pathways that could occur after release of DNA intothe cytoplasm. FIG. 12a shows a pathway similar to that seen in FIG. 8where the construct has been designed such that there is a single tRNAbinding site at the 3′ end of the DNA CNMAC. Priming and extension invivo by cellular mechanisms result in a single double-stranded DNAmolecule. FIG. 12b shows a pathway where the construct has been designedsuch that there are multiple tRNA binding sites at the 3′ end of theCNMAC. These can either be identical or different tRNA species can beused. Extension from a CNMAC with sequence for three tRNA primers (asshown in FIG. 12b) leads to the synthesis of a double-stranded DNAmolecule and two single-stranded DNA molecules. These latter twomolecules can be converted into double-stranded molecules if thesequence chosen for the ligand modified primer is also similar to a tRNAprimer sequence. When the construct is designed such that the pathwaywill be similar to that shown in FIG. 12a, the construct provide atranscript in which a) sequence J K represents a sequence complementaryto the ligand modified primer b) the sequence A B represents a sequencefor a CMV promoter c) the sequence C D E F represents a sequence forbiological function which will be expressed after transcription by theCMV promoter and d) sequence G H which is designed such that itscomplementary sequence will be a primer Binding site similar to the oneused by HIV to bind a cellular tRNA^(lys) as a primer for ReverseTranscriptase. When the construct is designed such that the pathway willbe similar to that shown in FIG. 12b, the construct provide a transcriptin which a) sequence J K represents a sequence complementary to theligand modified primer b) the sequence A represents a sequence for a CMVpromoter c) the sequence B represents a sequence for biological functionwhich will be expressed after transcription by the CMV promoter and d)and sequences C D, E F and G H represent sequences that arecomplementary to sequence will be primer Binding sites for tRNA's thatcan be used as primers. The major difference between the net result ofthe pathways shown in this example and previously described in Example 7and Example 8 is that the two latter examples depended upon the in vivopresence of Reverse Transcriptase whereas the present example providesthe Reverse Transcriptase activity in vitro prior to binding and uptakeinto target cells.

Example 10 Preparation of a Double-Stranded CHENAC Containing Moietieson Each Strand

[0402] A construct is made with the appropriate structure shown in FIG.13. Transcription is carried out in vitro by use of a T7 promoterdirecting the synthesis of the sequences of interest. The transcriptcontains a) sequence A B, which represents a sequence complementary to alactyl-LysylLysine modified DNA primer (prepared as describedpreviously), b) sequence C D which represents a CMV promoter fordirecting synthesis of a transcript in vivo, c) sequence E F whichrepresents a sequence for biological function which will be expressedafter transcription by the CMV promoter and d) sequence G H which isidentical to the sequence of a second modified primer that has fusogenicpeptides attached (prepared as described previously). In FIG. 10, thelactyl ligands are depicted by X X on the first primer and the fusogenicpeptides are shown as Z Z in the second primer. DNA is synthesized invitro by using the transcript as a template for Reverse Transcriptasewith the tri lactyl lysyl lysine modified DNA segment as a primer. Theresulting RNA/DNA double stranded molecule is treated with Rnase H toyield single stranded DNA. The second primer containing the fusogenicpeptides is then used as a primer to prepare the complimentary secondstrand of DNA.

[0403] This complex could be used either in vivo, ex vivo or in vitro tobind the DNA to a target cell through a ligand/receptor interaction. Theligand modified primer will promote uptake of the complex and afterendocytosis the fusogenic peptide modified primer will promote therelease of the DNA from the endosomes.

Example 11 A Bifunctional Binder Composed of a Bispecific Antibody

[0404] The methods of recombinant DNA are used to prepare a bispecificantibody with specificities for the CD4 protein of lymphocytes and formurine leukemia virus (FIG. 14). The antibody is prepared from murinemonoclonal antibodies according to the procedure of Staerz and Bevan(1985 Proc Natl Acad Sci USA 83; 1453) for the production of hybridhybridomas.

[0405] Antibody Modifications.

[0406] Hydrazine groups are introduced to antibodies in the carbohydratemoieties after oxidation with periodate or galactose oxidase andsubsequent reaction with hydrazine. When galactose oxidase is used forantibody oxidation, it is necessary to analyze for free galactose groupsas follows. The antibody is oxidized with galactose oxidase in thepresence of a peroxidase. At the end of the reaction the mixture isreacted with Lucifer Yellow CH (Aldrich) and passed through a G50column. If the flow through from the column fluoresces, this is anindication that the antibody contains free galactose residue and thatthe galactose oxidase can be used for antibody activation.

[0407] Ten mg antibody are dissolved in 1 ml of 0.1M acetate buffer, pH5.0, and oxidized with 1.0 umole NalO₄ at 4° C. for 30 minutes. Excessperiodate is removed by Sephadex G50 (Pharmacia) chromatography in 0.05Macetate buffer, pH 5.0. The protein fractions are combined and reactedwith 1.0 umole hydrazine acetate, pH 5.0, for 30 minutes at roomtemperature. The pH is raised to 9.0 with sodium carbonate and thecontents are cooled to 0° and 10 umoles sodium borohydrate are added inthree portions at ten minute intervals. The reduction is continued foran additional 60 minutes and the antibody is precipitated with 55%ammonium sulfate. After 2 hr at 0° C. the reaction mixture iscentrifuged for 30 minutes at 10,000×g. The pellet is dissolved in 1 mlacetate buffer, pH 5.5, and dialyzed in the cold against 0.1M acetatebuffer, pH 5.5.

[0408] One umole of 3-maleimidipropionic acid N-hydroxy-succinimideester is dissolved in 0.5 ml dimethylsulfoxide and added slowly to thedialysate and incubated for 30 minutes at room temperature. Excessmaleimide is removed by G50 chromatography and the combined antibodyfractions are reacted with the thiol containing ligand for 1 hr at roomtemperature at pH 6.5. Subsequently the conjugated antibody is separatedform the unreacted ligand by molecular sieving chromatography of theappropriate pore size.

[0409] Oligonucleotides synthesized with a thiol group at the 5′ end orthe thiol groups were added by reaction with an allylamine residue atthe 5′ or 3′ end of the nucleic acid with homocysteine thiolactone at pH9.0.

Example 12 A Bifunctional Binder Composed of an Antibody to the CD4 CellSurface Protein as the Domain for the Cell and a Single Stranded DNAMolecule as the Domain for the Nucleic Acid Component (FIG. 15)

[0410] A single stranded DNA molecule 120 bases in length and containinga 5′ terminal nucleotide modified by the addition of an allylamine groupis prepared chemically by the method of Cook et al. (1988 Nucleic AcidsRes 16;4077), and the allylamine residue is thiolated as in Example 11.The 70 bases at the 3′ end are complementary to the single strandedregion of Adeno Associate Virus DNA. The single stranded DNA is attachedto the F(ab′)₂ fragment as in Example 11 and they anneal to AdenoAssociated Virus as indicated in FIG. 15.

Example 13 A Binder Composed of a Bispecific Antibody (or of the F(ab′)₂Fragment of a Bispecific Antibody) Attached to a Single Stranded DNADomain for the Nucleic Acid Component (FIG. 16)

[0411] A bispecific antibody is prepared as described in Example 11 froma murine monoclonal antibody to CD34 cell surface protein and and amurine monoclonal antibody to adenovirus. The single stranded DNAmolecule described in Example 12 is attached to the bispecific antibody(or to the F(ab′)₂ fragment of the bispecific antibody) and annealed tothe adeno associated virus. An inactivated adenovirus is bound to theantibody (Cristiano et al. 1993 Proc Natl Acad Sci USA 90;2122: Curielet al. 1991 Proc Natl Acad Sci USA 88;8850) in order to facilitatecellular uptake of the complex.

Example 14 A Binder Composed of a Domain for Adeno Associated Virus DNA,a Domain for Binding to Liver Cells and an Inactivated Adenovirus (FIG.17)

[0412] Preparation of lactyl oligolysine 10 mer. Oligolysine issynthesized containing a cysteine residue at the carboxy terminus. Thethiol group is blocked with Eliman's reagent and the amino groups arereacted with a threefold excess of lactylisothiocyanate in 0.1Mbicarbonate buffer, pH 9.0, and 20% dimethylformamide for 2 hr at roomtemperature. The reaction mixture is chromatographed on a G50 column andthe lactyl-oligolysine fractions are combined and freeze dried. Thesolid is dissolved in 2 ml 1 mM dithiothreitol to unblock the protectedthiol group and chromatographed again on a G50, column to remove theexcess dithiothreitol and the liberated ElIman's reagent. All operationsare performed with argon saturated buffer to prevent thiol oxidation byair. The combined lactyl oligolysine fractions are combined and reactedimmediately with the maleimide derivatized antibody (see below) orproteins in a mixture with thiol containing nucleic acid as in Example12.

Example 15 An Antibody Binder with an Attached DNA with Domains forAdeno Associated Virus DNA and for Binding to Liver Cells (FIG. 18)

[0413] A single stranded DNA molecule 1000 bases in length and with a 5′terminal nucleotide containing a thiol group is synthesized chemicallyAllylamine groups are interspersed at 10 base intervals along the 50bases at the 5′ end of the molecule Cook et al.) and the 50 bases at the3′ end of the molecule are homologous to adenovirus associated virusDNA. After blocking the thiol groups, the lactyl groups are added asdescribed in Example 11. The thiol groups are then unmasked and thelactyl modified single stranded DNA is added to to a murine monoclonalantibody to adenovirus and it is annealed to adenovirus associated virusDNA as described in Example 12.

Example 16 Preparation of a Multimeric Antibody by Means of Nucleic AcidHybridization

[0414] (i) Preparation of Homopolymer

[0415] Oligo(dA) and oligo(dt) with an amine group at the 5′ end weresynthesized chemically. Longer molecules were prepared by using theamine-containing oligos as primers in a reaction with with Terminaltransferase and the appropriate dNTP precursors depicted as NA in FIG.19 and 20.

[0416] (ii) Preparation of Homopolymer Linker

[0417] 1,2 Diamino-4-Bromo-5-Hydroxycyclohexane was prepared accordingto U.S. Pat. No. 4,707,440 where the product of the (11-5) reaction wasreacted with N-Bromosuccinimide as in step (4-7) to yield compound I.(The various steps in this synthesis are shown in FIGS. 19 and 20).Compound I was reacted with a 5-fold excess of dithiothreitol at 90° C.,pH 8.0 in argon atmosphere for 2 hours. The reaction mixture wasacidified to pH 1.0 and the excess of dithiothreitol was removed byperoxide-free ether until no thiol was detected in the ether phase. Theaqueous phase which contains Compound II was used for the next step.

[0418] (iii) Attachment of Linker to Homopolymer

[0419] The 5′ amino group of the nucleic acid was reacted with3-maleimidopropionic acid N-hydroxy succinimide ester in 0.2 M sodiumbicarbonate buffer pH 7.8 and 0.7 M lithium chloride 30% dimethylformamide for 40 minutes at 25° C. The pH of the mixture was brought to5.5 with 2.0 M acetic acid and the excess active ester was removed byextraction with n-butanol. The product Compound III was precipitatedwith 4 volumes ethanol for 2 hours at −70° C. It was centrifuged and thepellet was dissolved in 0.7 M lithium chloride and reacted immediatelywith excess Compound II at pH 6.0 for 30 minutes at room temperature toyield Compound IV; it was separated from excess of Compound If byethanol precipitation as in the previous step. Compound IV was reactedwith excess 3-maleimidopropionic acid N-hydroxy succinimide ester (asdescribed in the preparation of Compound III) to yield Compound V. Theproduct was precipitated twice with 4 volumes ethanol and stored as apellet at −70° C. until used.

[0420] (iv) Preparation of Antibody

[0421] Fab′-SH fragments were prepared by reduction of F(ab′)₂ antibodywith 0.5 M dithiothreitol at pH 7.5 (Taizo Nitta, Hideo Yagita,Takachika Azuma, Kiyoshi Sato and Ko Okumura Eur J. Immunol 1989 19:1437-1441) under argon atmosphere. The pH was lowered to 6.0 and theantibody was separated from dithiothreitol by G50 chromatography usingfully deaerated buffer under argon atmosphere to prevent oxidation toF(ab′)₂

[0422] (v) Attachment of Homopolymer to Antibody Fragments

[0423] The protein fractions from step (iv) were combined and reactedwith Compound V (FIG. 20) from step (iii) in a 2:1 ratio to formCompound VI, always under argon atmosphere and in the presence of 2 mMEDTA to prevent nuclease action. After overnight incubation at 4° C.,Ethylmaleimide was added to the reaction mixture to block any free thiolresidues and the protein was precipitated with ammonium sulfate (60% ofsaturation). The pellet was dissolved in minimum amount tris-HCl buffer,pH 7.8 and chromatographed in a G100 column to separate the conjugatefrom the reaction products.

[0424] (vi) Annealing of Homopolymers to Obtain Antibody Multimers

[0425] Annealing is done 0.2M NaCl, 0.05M Tris HCl (pH 7.8), 1 mM EDTA.FIG. 21 shows the overall outline of the process. In the last step shownin FIG. 21, (a) shows an example where both the A homopolymer and the Thomopolymer are short enough that there is essentially only one of eachtype of molecule binding together in a 1:1 ratio. The (b) diagram showsthe situation where the A homopolymer was synthesized such that its muchlonger than the T homopolymer; in this situation, larger numbers ofantibodies can be linked together into complexes.

Example 17 Preparation of a Multimeric Insulin by Means of Nucleic AcidHybridization

[0426] Oligo T with a primary amino group (prepared as describedearlier) is reacted in 0.7M LiCl, 0.1 M sodium bicarbonate buffer, pH7.8 and 30% dimethyl formamide with a 3-fold excess of suberic acid bis(N-hydroxysuccinimide) ester for 15 minutes at room temperature. The pHwas then lowered to 5.0 by the addition of 2M acetic acid and the excessof active ester was extracted twice with n-butanol. The nucleic acid wasprecipitated with 4 volumes ethanol at −70° C. and the pellet aftercentrifugation was dissolved in cold 0.7 M LiCl in 0.1 M sodiumbicarbonate solution (pH 7.8), solid insulin was added in 1:1.2 ratioand the conjugation was allowed to take place at 4° C. overnight. Theproduct is separated from the reactants by molecular sievingchromatography on G75 columns. A multimeric complex is formed by thehybridization of the T-tailed insulin molecules with a Poly A binder asdescribed earlier. The steps in this Example are shown in FIG. 22.

Example 18 Preparation of a Multimeric Insulin by Means of Nucleic AcidHybridization Through Specific Discrete Sequences

[0427] A group of nucleic acid sequences are selected from the knownsequence of the single-stranded form of bacteriophage M13. These arethen artificially synthesized such that they have a primary amino groupon the nucleotide at the 5′ end, the oligomers are individuallyactivated and attached to insulin molecules as described in Example 17.A mixture is made of each of the oligomerlinsulin complexes and mixedwith M13 DNA derived from phage particles (the + strand). The productwas separated from the reactants by molecula sieving chromatography. Thesteps in this Example are shown in FIG. 23.

Example 19 Synthesis of a Eukaryotic Vector that Expresses T7 RNAPolymerase as Well as Antisense Sequences Directed by a T7 Promoter

[0428] (A) Intron and Intron Insertion Site

[0429] The SV40 small T intron has been utilised in a number of DNAvectors and it has been chosen for this particular example due to itssmall size and the presence of stop codons in all three reading frames.The consensus sequences for splice donors and acceptors are partiallymade up by exon sequences as well as intron sequences. A computer searchusing the MacDNASIS program (Hitachi, Inc.) allowed the identificationof 19 different sites within the T7 RNA polymerase coding sequence(Mount, 1982 Nucleic Acids Research 10,459) that contain the sequence(C/A)AGG, which as described earlier is a consensus sequence for apost-splice junction. Any of these sites should be suitable for theintron insertion site, but for this example, a T7 site was chosen thatclosely resembled some of the flanking exon sequences of the SV40intron. FIG. 24 shows the sequences surrounding this site in the T7 RNApolymerase gene sequence and the subsequent insertion of the SV40 virusintron into this site. FIG. 24 also shows the mRNA made from this fusionand the subsequent splicing out of the Intron sequence to reconstitutethe normal T7 coding sequence.

[0430] (B) Fusion of Intron Sequences into the T7 Coding Sequences

[0431] A method for introduction of the intron and production of avector that contains the interrupted T7 RNA Polymerase as well assequences directed from a T7 promoter is given in FIG. 25. As shown inFIG. 25, the creation of this construct can be accomplished by PCRamplifications of each segment of the T7 RNA polymerase gene (left andright of the intended intron insertion site) and PCR amplification of aeucaryotic intron. These pieces are joined together using cloning stepsdescribed below. It has previously been shown that PCR products can befused together by a technique referred to as “Splicing by OverlapExtension” (SOE) to generate precisely joined fragments without extrasequences being added (Horton et al 1990 BioTechniques B: 528; Horton etal., 1989 Gene 77: 61). However, in addition to the PCR reactions neededto create the different segments, the SOE method involves the use ofthese PCR products as primers in a secondary PCR reaction to fuse thesegments. For fusions of multiple segments there would be a series ofsequential PCR reactions to be carried out. Even with thermostable DNApolymerases chosen for a lower error frequency, the synthesis of thefinal product will require that some sequences be subject to severalmulticycle amplification steps thereby leading to an increased chance ofundesirable mutations in the final product. For this reason, theinventors of the technique advised sequencing the final product toinsure that the desired product was obtained (Horton et al., 1990). Inthe present example, a method was used that requires only an initialround of PCR amplification to create each segment followed by ligationof the segments together to form the final fused product. Fusions of thegene segments and intron to form the appropriate product were carriedout by addition of restriction enzyme sequences onto the 5′ end of thePCR primers to allow the production of “sticky ends” (Scharf at al.,1986 Science 233: 1076). To give the precisely defined end points forthis fusion, restriction enzymes (Bsa I and Bsm B1) that recognizenon-palindromic sequences and cut outside of their recognition sequenceto leave a single stranded tail with arbitrary definition were used.This method allows joining of sequences at any point chosen by the userby the appropriate design of the PCR primers.

[0432] (C) Synthesis of the Individual Segments Used for the Fusion

[0433] The T7 RNA polymerase is encoded by bases 3171-5822 in the T7genome (Dunn and Studier, 1983 J. Mol. Biol. 166: 477) and this sequenceis available in Genbank as Accession #'s V01146, J02518 or X00411. Basedupon this information, six different oligos were synthesized. The use ofthese oligo's and their sequences are given in FIG. 26. TSP 1 and TSP 2were annealed together by a 12 bp complimentary sequence and extended toform a completely double-stranded DNA molecule (FIG. 27). Conditionswere as follows: 150 pM of TSP 1, 150 pM of TSP2, 1×NEB Buffer #2 (NewEngland Biolabs, Inc.), 200 uM dNTP and 13 units of Sequenase v2.0 (U.S.Biochemicals, Inc) for 75 minutes at 37° C. TSP 3 and 4 were used in aPCR reaction (Saiki et al. 1985 Science 230, 1350)) with T7 genomic DNAas a template to synthesize the “Left” fragment. Reagent conditions wereas follows: 100 ul volume containing 100 ng T7 template (Sigma ChemicalCo.), 1 uM TSP 3, 1 uM TSP 4, 1 mM MgCl₂, 1×PCR buffer, 250 uM dNTP, 2.5units of Taq DNA Polymerase. Temperature cycling conditions were: 16cycles of (1) 50 seconds at 94° C. (2) 25 seconds at 50° C. and (3) 3minutes at 72° C. The same conditions were used to form the “Right” endfragment with Oligomers TSP-5 and TSP-6 except that due to the length(over 2 kb) of the expected product, 2.5 units of Taq Extender(Stratagene, Inc) was added and the Taq Extender buffer substituted forthe normal PCR buffer. INT-1 and INT-2 were used together in a PCRreaction to form the Intron piece. Conditions were the same as thoseused for synthesizing the “Left” fragment of T7, except that a clone ofSV40 was used as the template and due to the smaller size of theamplicon, the cycle conditions were only 1′ at 72° C. for the extensiontime. FIG. 27 shows the synthesis of the short double stranded piece ofDNA made by extension of oligo's TSP 1 and TSP 2 and its combinationwith the left end of the TSP 3/TSP 4 PCR product to generate thecomplete (NLS+) T7 RNA polymerase. The resultant nucleic and amino acidsequences are given in FIG. 28 for the construct given in this exampleas well as the normal wild type T7 RNA polymerase sequences.

[0434] Thus, the modifications carried out at the 5′ end during thisconstruction process were:

[0435] a) The sequence around the ATG start codon was changed to give aKozak consensus sequence (Kozak 1984 Cell 44: 283) to increaseefficiency of translation of the gene product. This change hadpreviously been introduced into the T7 RNA polymerase coding sequence.

[0436] b) The fusion of the TSP1/TSP2 extension product to the TSP3/TSP4PCR introduces a 9 amino acid insertion between bases 10 and 11 in thenormal T7 RNA polymerase protein sequence. This sequence has previouslybeen shown to be a signal for transportation to the nuclease byKalderone et al. (1984 Cell 39: 499) and had been introduced into T7 RNApolymerase by Lieber et al., (1989) as a substitute for the first 10amino acids and inserted into an artificially created EcoR1 site by Dunnet al., (1988). The method used in this Example to introduce the NuclearLocalisation Signal (NLS) was designed to minimize perturbations to thenormal structure of the protein. The codons for the amino acids codingfor the NLS are indicated as larger type size in FIG. 28

[0437] (D) Combination of Pieces to Form the Final Construct of the T7RNA Polymerase Gene in a Eucaryotic Expression Vector

[0438]FIG. 29 shows the various steps used for this process. For ease ofuse, each of the three pieces {PCR #1, PCR #2 and PCR #3} was clonedinto a plasmid vector (PCR II) using the TA cloning kit and followingthe manufacturer's instructions (Invitrogen, Inc.).

[0439] PCR #1 (the left end of the T7 RNA polymerase) was cloned intoPCR II to create pL-1. This construct was then digested with Bsm1 andSpe I to excise out the PCR product and the TSP1/TSP2 Extension product(shown in detail in FIG. 27) was digested with Eco R1 and Bsa I. Due tothe design of the primers, the single-stranded tails created by BsmB1and Bsa I are complimentary to each other and ligation of these piecesforms a single piece with an EcoR1 tail at one end and a Spe I tail atthe other end. Digestion of the M13 vector, mp18, with EcoR1 and Xba Iallows insertion of the EcoR1/Spe I piece to form pL-2.

[0440] PCR #2 (the SV40 Intron) was cloned into PCR II to form pINT-1.This construct was digested with EcoR1 and Spe I and transferred intothe M13 vector (mp18 digested with EcoR1 and Xba I) to form pINT-2.

[0441] PCR #3 (the right end of the T7 RNA polymerase) was cloned intoPCR II to create pR-1. This construct was digested with Eco R1 and Spe Iand then self-ligated to form pR-2. This step was added to eliminateextra EcoR1 and Spe I sites present in pR-1.

[0442] As described in FIG. 25, the elements in pL-2, pINT-2 and pR-2are fused together to form the complete intron-containing T7 RNApolymerase. This was accomplished by digestion of pL-2 with BsmB1 andBsa I; pINT-2 with BsmB1; and pR-2 with BsaI and Spe I. Ligation ofthese three inserts together forms a single fragment that has one endcompatible with a Hind III end and the other end compatible with Spe I.This fragment was cloned in the same step into pRc/RSV (from Invitrogen,Inc.) that had been previously digested with Hind III and Spe I. Asshown in FIG. 29, this final produet is pINT-3. This particulareucaryotic vector was chosen since it had beeen shown previously thatthe RSV promoter is especially active in hematopoietic cell lines. Also,the ligation of the Hind III end from pRcRSV to the end created from theBsmB1 digestion of pL-2, does not reconstitute the Hind III site inpINT-3, the final product.

[0443] E) Antisense Sequences

[0444] Three different targets in the HIV genome were chosen as testtargets for Antisense: (A) the 5′ common leader, (B) the coding sequencefor Tat/Rev and (C) the splice acceptor site for Tat/Rev. Antisense to(A) was derived from a paper by Joshi et al. (1991 J. Virol. 65,5534);Antisense to (B) was taken from Szakiel et al. (1990 Biochem Biophys ResComm 169, 213) and the Antisense to (C) was designed by us. Thesequences of the oligo's and their locations in the HIV genome are givenin FIG. 30. Each oligo was designed such that annealing of a pair ofoligo's gives a double-stranded molecule with “sticky ends” that arecompatible with a Bam H1 site. The oligo's were also designed such thatafter insertion into a Bam H1 site, only one end of the molecule wouldregenerate the Bam H1 site, thus orientation of the molecule couldeasily be ascertained. The resultant clones were termed pTS-A, pTS-B andpTS-C for the anti-HIV sequences A, B and C respectively.

[0445] F) Cloning of T7 Terminator

[0446] The sequence for termination of transcription by the T7 RNApolymerase is encoded by a sequence between the end of the gene 10bprotein at base number 24,159 and the start codon of the gene 11 productat base number 24,227 in the T7 genome (Dunn and Studier 1983 J. Mol.Biol. 166, 477) Genbank Accession #'s V01146, J02518 or X00411. Basedupon this information, TER-1 and TER-2 were synthesized (Sequences givenin FIG. 30) and used in a PCR amplification reaction to obtain adouble-stranded 138 bp piece that contained the T7 sequences from 24,108to 24,228 with an Xba I site added at one end and a Pst 1 site added tothe other. The reagent conditions for amplification were as describedfor the TSP3/TSP4 reaction but the temperature cycling conditions were:16 cycles of (1) 50 seconds at 94° C. (2) 25 seconds at 50° C. and (3) 1minute at 72° C. As shown in FIG. 30, the terminator piece was clonedinto the PCR II vector and then after XbaI/Pst I digestion it wastransferred into an M13 vector.

[0447] G) Creation of T7 Driven Antisense Transcription Units

[0448] The clones containing Antisense sequences (pTS-A, pTS-B andpTS-C) were digested with Eco R1 and Pst I while the clone containingthe T7 terminator (pTER-2) was digested with Xba and Pst I. These wereligated together with pIBI 30 (IBI, Inc.) that had been digested withEco R1 and Pst I to form the AntiSense transcription units shown in FIG.30 which have Antisense sequences transcribed from a T7 promoter andthen terminated by a T7 terminator. The resultant clones were termedpTS-A1, pTS-B1 and pTS-C1 for the anti-HIV sequences A, B and Crespectively.

[0449] H) Transfer of Antisense Transcription Units into pINT-3

[0450] By the nature of the present invention, the T7 driven AntisenseTranscriptions units can be transferred into pINT-3 to make a singleconstruct T7 polymerase/promoter construct. This was accomplished bycreating an M13 phage vector LIT ø-2 by transferring the polylinker fromthe plasmid vector LIT-38 (New England Biolabs, Inc.) by digestion withSpe I and SphI and ligating the polylinker insert into mp18 that hadbeen digested with Xba I and Sph I. This and subsequent steps are shownin FIG. 31. Clones pTS-A1, pTS-B1 and pTS-C1 which contain T7 directedAntisense sequences were digested with EcoRV and Pst I. They were thenligated to the LIT ø-2 vector which had also been digested with Eco RVand PstI. The resultant clones are phage vectors that contain T7directed Antisense sequences and were termed pTS-A2, pTS-B2 and pTS-C2respectfully. These clones were digested with Nhe I and Bsp 120 I andligated to the pINT-3 vector (from FIG. 29) that had previously beendigested with Spe I and Not I. the resultant clones pRT-A, pRT-B andpRT-C contain the coding sequence for the T7 RNA polymerase driven bythe RSV promoter and with an SV40 intron sequence that will be splicedout to form a functional polymerase enzyme and in addition eachconstruct contains an HIV Antisense sequence driven by a T7 promoter andterminated by a T7 terminator.

Example 20 Expression of a Protein made from T7 Directed TranscriptsDerived from a Single Construct that also Expresses the T7 RNAPolymerase

[0451] The pINT-3 vector used in the previous example can be modifiedfor use as an expression vector for T7 directed protein synthesis. Forthis purpose, the pINT-3 vector needs has a T7 promoter, a T7 terminatorand a polylinker in between. The optimal site for the placement of thesemoities is after the poly A signal for the T7 RNA polymerase in pINT-3where there is an Xho I and a Bam H1 site. Since there are also otherXho I and Bam H1 sites within the vector, manipulations of thisparticular segment can only be done if the small segment containing thisarea is separated out, the appropriate nucleic acids introduced inbetween the Xho I and Bam H 1 sites and then the segment replaced backin. The steps used for the creation of this construct are shown in FIGS.32 and 33.

[0452] a) Introduction of Polylinker

[0453] The segment containing the Xho/Bam H1 insertion site was derivedfrom the plasmid pRC/RSV, which was the parent of pINT-3. This was doneby digesting pRC/RSV with XbaI and Xma I and transferring theappropriate fragment into the plasmid pUC18 (NewEngland Biolabs, Inc.)previously digested with Xba I and Xma I to obtain the vector pEXP-1.This in turn was digested with Xho I and Bam H1 and then a polylinkerwas inserted by ligation with oligomers PL-1 and PL-2 (Sequences areshown in FIG. 32). The resultant plasmid was named pEXP-2 and therestriction sites contained with the new polylinker are shown in FIG.32.

[0454] b) Introduction of T7 Promoter and T7 Terminator

[0455] A promoter was inserted into pEXP-2 by digestion with Nco I andBam H1 followed by ligation with oligomers TPR-1 and TPR-2 (Sequencesare shown in FIG. B-10) to create pEXP-3. The normal T7 promoterconsensus sequence (Dunn and Studier, 1983) was not used since it hasbeen shown that it can function as a eucaryotic promoter in some celllines (Sandig et al., 1993 Gene 131;255) and a sequence derived fromLieber et al. (1993) was substituted since this equence still functionswell in the presence of T7 RNA Polymerase but remains silent in itsabsence. The vector pEXP-3 was digested with Spe I and Pst and ligatedto the T7 terminator fragment derived from the pTER-1 constructdescribed in the previous example in order to create the vector pEXP-4.The Xba/Xma segment has now been modified to contain the T7 terminator,a short polylinker and the T7 terminator. It was substituted for theunmodifed segment in pINT-3 by Xba I/Xma I digestion of pINT-3 andPEXP-4 followed by ligation as shown in FIG. 33 thus creating the vectorpINT-4.

[0456] c) Introduction of a Protein Coding Sequence into the New T7Expression Vector

[0457] The gene coding for the complete lac Z sequence was obtained frompZeoSVLacZ (Invitrogen, Inc.) by digestion with Age I and Cla I. Thiswas then ligated into pINT-4 that had been previously digested with BspE1 and ClaI to create pINT-LacZ (not shown). After introduction into aeucaryotic cell, the RSV promoter directs the synthesis of the T7 RNApolymerase which in turn acts upon the T7 promoter to synthexizeB-galactosidase.

Example 21 A Primary Nucleic Acid Construct that Propagates ProductionCenters for the Production of Produces Single-Stranded Antisense

[0458] A Primary Nucleic Acid Construct is described as shown in FIG. 34and 35 whereby, subsequent to introduction into a cell, a series ofevents, including self priming, multiple priming and Rnase H and reversetrasncriptase activities, leads to the production of single stranded DNAantisense molecules. In this case a Nucleic Acid Construct createsmultiple copies of a Production Center, an RNA transcript with hairpinstructure with a discrete 3′ end (structure 34a, FIG. 34). In thepresence of reverse transcriptase self priming occurs by the 3′ end ofthe hairpin acting as primer to extend to the 5′ end of the moleculeresulting in a hairpin structure composed of both DNA and RNA (structure34b). By a multiple priming process, Rnase H, either as part of theviral reverse transcriptase or from the Inherent Cellular Systems,starts degradation of the RNA bound to the DNA. Degradation can becomplete if there is enough Rnase H activity, or if the reversetranscriptase activity is high enough, the initiation of RNA degradationprovides RNA fragments that serve as primers for extension using the DNAportion as a template. In the former case the net result of thedegradation by RNase H is a single-stranded DNA molecule with a doublestranded 5′ RNA terminus (structure 34c); in the latter case (structure34d), the priming event results in a) the Production of a series ofmolecules such as 34f and 349, the length of the single-stranded DNAportion depending upon the site of the priming initiation event and b)the propagation of Production Centers such as structure 34e. Structure34g could act as a biological modifier if, for example, the sequencesrepresented as the Z single stranded DNA region were antisensesequences. Through the activity of RNase H and reverse transcriptase,structure 34e would be processed further to produce single stranded DNAmolecules (structures 351h, 35i and 35j, FIG. 35). which could act asantisense DNA if the sequences X′, Y′, Z′ were designed with thatpurpose. The Production of antisense DNA molecules according to thisinvention represents the first demonstration of the method for theintracellular synthesis of antisense DNA.

Example 22 A Primary Nucleic Acid Construct that Propagates an RNAProduction Center that is Reverse Transcribed to Create DNA ProductionCenters Capable of DirectingTranscription

[0459] In this example, the same processes of self priming and multiplepriming described in the Example 21 occur with the propagation of singlestranded DNA hairpin structures (FIG. 36). As in Example 21, structures36b, 36c and 36d (FIG. 36) act as Producton Centers for the Productionof single stranded RNA. In this case this represents an amplificationevent since reverse transcriptase and RnaseH convert a single ProductionCenter (36a), into a double stranded DNA Production Centers (36b, 36cand 36d) which can direct the Production of multiple single stranded RNAmolecules.

Example 23 A Primary Nucleic Acid Construct which Propagates a DoubleHairpin Production Center for the Production of Single Stranded RNA

[0460] In this example, a double stranded DNA Primary Nucleic AcidConstruct (structure 37a, FIG. 37) has been designed such that a singlestranded Production Center, propagated from it, forms hairpin structuresat the 5′ and 3′ ends. Extension by self priming from the 3′ endfollowed by further steps catalyzed by RnaseH and reverse trancriptaseresult in the propagation of a double-stranded DNA molecule with singlestranded hairpin ends (structure 38b, FIG. 38). This can be furtherprocesesed, by the action of DNA ligase, to form a covalently closedmolecule (38c) or by the action of reverse transcriptase to form alarger linear molecule (38d). The presence of promoters and codingsequences in these Production Centers provides for Production of singlestranded RNA. As seen above in Example 22, this is an amplificationevent since each Production Center producing RNA transcripts was itselfderived from a single transcript.

Example 24 A Nucleic Acid Construct which Propagates a Production Centercapabale of Inducible Call Destruction

[0461] In this example (FIG. 39) provides for the production a singlestranded nucleic acid as a result of the introduction into cell of aninherent cellular system. In this case, the events leading to thePropagation of a Production Center (structure 39b) are brought about bythe presence of Reverse Transcriptase. Here, the single stranded nucleicacid product of a Production Center is mRNA which can be translated toproduce a lethal product, diphtheria toxin, resulting in a reversetranscriptase dependent cytocidal event. Elimination of low levelsynthesis of a toxic gene product such as diphtheria toxin in theabsense of viral infection TAT activation (as was observed by Harrisonet al.) is accomplished by the use an intron artificially inserted intothe non-coding strand (39a) of the segment coding for the toxin. In thisway, transcription of the toxin sequence will not produce an activeproduct. Production of active toxin only occurs when the antisensetranscript is spliced and used as a template for Reverse Transcriptase.

[0462] The result of Rnase H and reverse transcriptase mediatedactivities is a double stranded DNA Production Center (39c) that has atemplate for the toxin and which has the intron sequences removed. As afurther refinement, the promoter sequence in the double-stranded DNAProduction Center (region designated as ABC in structure 39b) can be anHIV LTR. In this case Production of the toxin would be dependent upontwo events that should be provided by viral infection.

Example 25 Use of tRNA Primers to Create a Double-Stranded DNAProduction Center for Production of Single Stranded RNA

[0463] This example utilizes the presence of primer binding sites in asingle stranded RNA Production Center for the Propagation of adouble-stranded DNA Production Center. In this way, sequences derivedfrom the Primer Binding Sites of retroviruses, such as the HIV primerbinding site which utilizes lysyl tRNA as a primer, can be inserted nearthe termini (regions designated X and Y) in the RNA Production Center(FIG. 40, structures 40b and 40c) for the priming of DNA synthesis toform double stranded DNA Production Centers. The resultant ProductionCenters, such as structure 40d, are double stranded DNA molecules butcan function as described previously to produce single stranded RNAwhich either can be utilized as anti-sense nucleic acid or which can betranslated to produce a protein.

Example 26 Construction of Plasmids with Anti-sense Segments Introducedinto the Transcript Region of the U1 Gene

[0464] The overall process used in this example is depicted in FIG. 41.The gene for U1 is present in the plasmid pHSD-4 (Manser and Gesteland1982 Cell 29;257). Three different pairs of deoxyoligonucleotides weresynthesized and the sequences are given in FIG. 42. The pairs werehybridized to form double stranded molecules with single strandedoverhangs to form sites compatible with the BcI/Bsp ends in the plasmid.The Bcl/Bsp ends in the plasmid remain after removal of the 49 basesequence from the U1 coding sequence. When each sequence is insertedinto and expressed from the U1 coding region of pHSd-4 U1 it will appearas an antisense RNA sequence to a region of the HIV gemome.

[0465] After digestion with BcI 1 and Bsp E1, a 49 base pair segment iseliminated from the U1 transcript portion of the gene. The oligo pairshave been designed to form sticky ends compatible with the BcI/Bsp endsin the plasmid. Ligation of each of the pairs of Oligo's (HVA-1+HVA-2,HVB-1 +HVB-2 and HVC-1+HVC-2) created pDU 1-A with an insertion of 72bp, pDU1-B with an inserion of 66 bp and pDU1-C with an insertion of 65bp. As a control, two oligomers (HVD-1 and HVD-2) with sequencesunrelated to HIV were also inserted into the U1 operon to create pDU1which contains an insertion of 61 bp.

[0466] To allow for selection of transformants after introduction ofthese chimeric U1 genes, the Neomycin resistance gene was introduced bydigestion of pGK-neo (McBurney et al. 1991 Nucleic Acids Research19;5755) with Hind III and Sma I and ligation into the pDUI series ofplasmids previously digested with Hind III and Hinc II to create thepNDU1 series (pNDU1-A, pNDU1-B, pNDU1-C and pNU1-D).

[0467] As described earlier, the design of the cloning method shouldallow the insertion of novel sequences that would still allow theutilisation of signals provided by the U1 transcript for nuclearlocalisation of Anti-sense sequences. To test whether the insertion ofthe sequences described above resulted in unintended changes in the U1region responsible for re-importation of the U1 transcripts a computeranalysis was done to compare the predicted structures for the normal U1and the chimeric novel molecules using the MacDNASIS program (Hitachi,Inc.). In FIG. 43 it can be seen that despite changes in the 5′ end(where the new sequences have been introduced) loops III and IV as wellas the Sm region remain undisturbed.

Example 27 Construction of a Multi-Cassette Construct which ExpressesThree Antisense Sequences as Part of U1 snRNA

[0468] The various steps used in this example are depicted in FIG. 44.The various constructs used in this example, pDU1(A), pDU1(B), pDU1(C)and pGK-neo were described in Example 26 of this patent. The plasmidpDU1(B) with the “B” anti-sense embedded within the U1 transcript wasdigested with Sma I and Hind III. The segment containing the U1 operonwith the “A” anti-sense was released by digestion of pDU1 (A) with HincII and Hind III and ligated into the pDU1(B) plasmid to create pDU1(A,B)which contains two separate operons for the “A” and “B” anti-sensesequences. This construct was then digested with Sma I and Hind III (torelease the double operon) and ligated into pDU1(C), containing the U1operon with the “C” anti-sense, that had previously been digested withHinc II and Hind III. The resultant construct, pDU1(A,B,C) containsthree separate operons containing the “A”, “B” and “C” anti-sensesequences. To allow selection for the presence of this construct after atransfection step, the segment containing Neomycin resistance wasexcised from the vector pGK-neo by digestion with Hind III and Sma I andligated into the pDU1(A,B,C) construct to create pNDU1(A,B,C). Theordering of the three operons in the pDU1(A,B,C) and pNDU1(A,B,C)constructs is given in FIG. 46.

Example 28 Construction of an Antisense Expressing Multi-CassetteConstruct Containing Three T7 RNA Promoters

[0469] The various steps used in this example are depicted in FIG. 45.The polylinker from plasmid LIT 28 (New England Biolabs, Inc.) wastransferred into an M13 vector by digestion of the plasmid with BgII andHind III and then ligating it with mp18 (New England Biolabs, Inc.)previously digested with Bam H1 and Hind III to create the phage vectorLIT ø1. The plasmid pTS-B (described in Example 19) containing a T7promoter, the “B” Anti-Sense sequence and the T7 terminator, wasdigested with EcoRV and Hind III and then ligated to LIT ø1 previouslydigested with EcoRV and Hind III to create TOP 302, a phage vector withthe “B” Anti-sense T7 operon.

[0470] The polylinker from plasmid LIT 38 (New England Biolabs, Inc.)was transferred into an M13 vector by digestion of the plasmid with SpeI and Sph I and then ligating it with mp18 previously digested with XbaI and Sph I to create the phage vector LIT ø2. The plasmid pTS-A(Example 19) containing a T7 promoter, the “A” anti-sense sequence andthe T7 terminator, was digested with EcoRV and Pst I and then ligated toLIT ø2 previously digested with EcoRV and Pst I to create TOP 414, aphage vector with the A Anti-sense T7 operon. The T7 operons in TOP 302and TOP 414 were joined together by digestion of TOP 302 with Mlu I andBsi W1 and ligating it to TOP 414 previously digested with Mlu I and BsrG1 to form TOP 501, a phage vector which has both the “A” Anti-Sense T7operon and the “B” Anti-Sense T7 operon.

[0471] The plasmid pTS-C (described in Example 19) containing a T7promoter, the “C” anti-sense sequence and the T7 terminator, wasdigested with Sph I and Hind III. TOP 501 was then digested with SphIand Hind III and ligated to pTS-C2 to create TRI 101 which has the “A”Anti-Sense T7 operon, the “B” Anti-Sense T7 operon and the “C”Anti-Sense T7 operon in a single construct. The ordering of the threeoperons in the TRI 101 construct is given in FIG. 46. Co-transfection ofthis construct with a vector that expresses T7 RNA polymerse (The Introncontaining T7 RNA Polymerase described in Example 19 could be used forthis purpose) allows the in vivo production of all three Anti-Sensetranscripts.

Example 29 Construction of an Antisense Expressing Multi-CassetteConstruct Containing Three T7 RNA Promoters and an Intron-Containing T7RNA Polymerase Gene

[0472] Although the preceding example utilises the common method ofexpressing T7 directed transcripts by means of cotransfection with aconstruct with the RNA polymerase and a second construct with a T7promoter, an application of the current invention describes a method ofcarrying both entities (polymerase and promoter) on the same construct.The present example is an illustration of a single construct thatcontains the T7 RNA polymerase as well as multiple operons of T7 drivenAnti-Sense transcripts. The various steps used in this example aredepicted in FIG. 47. The plasmid pTS-C (described above) was digestedwith EcoRV and Pst I and ligated into the M13 vector LIT ø2 (describedabove) which had previously been digested with EcoRV and Pst I, tocreate the TOP 601 which is a phage vector with the “C” Anti-Sense T7operon. As described earlier, the construct pINT-3 contains the T7 RNAPolymerase with an SV40 intron inserted within the coding region; ineucaryotic cells there is expression by an RSV promoter followed byexcision of the intron by means of the normal splicing machinery of thecell. To insert the T7 Anti-sense operons, it was digested with Spe Iand Not I. The T7 Anti-sense operons were inserted as a triple insert bythe simultaneous ligation of the Spe/Not pINT-3 DNA with TOP 601previously digested with Pst I and Nhe I, TOP 302 previously digestedwith Mlu I and Nsi I and TOP 414 previously ligated with Bpu 120 1 andMlu I. The resultant clone as well as a diagram of the positions of thedifferent Anti-sense operons is shown in FIG. 47.

Example 30 Testing the Anti HIV U1 Constructs in Cells: Inhibition ofVirus Growth

[0473] a) Creation of Stable Transformed Cell Lines

[0474] U937 cells (Laurence, et al., 1991, J. Virol. 65: 214-219) weretransformed with the various U1 constructs described above usingLipofectin (BRL Inc.) and following the manufacturer's suggestedprotocol. After transformation, the cultured cells were divided into 2portions. One portion was used to obtain individual clones while theother portion was used to obtain a population of pooled clones. Toobtain the individual clones, aliquots of 1×10⁴ cells were seeded intoseparate chambers in 96 well tissue culture plates and stabletransformants were selected by growth in DMEM (Gibco and BRL) mediumsupplemented with 10% fetal bovine serum (heat inactivated) (Gibco andBRL) in the presence of 600 microgram\ml G418 (Gibco and BRL). TheG418-containing medium was replaced every 3 to 4 days, and after 3 weeksof incubation, drug resistant cells were removed from individual wellsby aspiration and expanded by growth in culture dishes. To obtain thepopulation of pooled clones, 1×10⁶ cells were seeded into T-25 flasks(Corning) and grown in the presence of G418.

[0475] b) Characterization of Cell Lines

[0476] RNA was isolated from either resistant clones or resistant pooledclones using hot phenol extraction (Soeiro and Darnell, 1969, J. MolBiol 44: 551-562). This RNA was used in a dot blot analysis using theprotocol accompanying the Genius System (Boehringer Mannheim). The probeused in this analysis was a riboprobe made from a clone of the threeinserts (A, B, and C) in pBlueScript (Stratagene) cloned into the XmaIand BamH1 site. This clone produced insert RNA of the sense orientation.The results of this analysis showed that all cell populations that hadbeen transformed by the U1 clone and that had demonstrated resistance toG418 that were tested expressed the antisense insert RNA. Comparable dotblot analyses were performed using RNA from the parental line U937 aswell as yeast RNA (Boehringer Mannheim.) These dots showed no evidenceof the antisense insert RNA. The antisense RNA synthesized in vitrousing the clones pBlueScript 12, pBlueScript 34, pBlueScript 56 andpBlueScript 78, described above, showed positive hybridization using thesense probe described in this paragraph. From this we conclude thatthose transformed cell populations that were tested were indeedexpressing antisense RNA from the HIV virus sequence.

[0477] c) HIV Challenge Experiment Number 1

[0478] 0.5×10⁶ cells of the pooled clones transformed by the triple U1construct were incubated with HIV virus at a multiplicity of 0.1 5 pfuof the virus per cell in the presence of 2 μg/ml of polybrene for 2hours at 37° C. using the procedure of Laurence et al. (1991 J. Virol.65: 214-219). The cells were then washed, resuspended in 1 ml of culturemedium (RPMI 1640+10% fetal bovine serum, Flow Labs) and plated induplicate (0.5 ml per well.) One-half of the culture medium was removedand replaced with fresh medium every 3-4 days. 6 days post infection,samples of these cells were tested for the extent of infection by HIVvirus using a p24 ELISA antigen capture following the protocol of themanufacturer (DuPont). The control cultures for this experiment werecells transformed by clones not containing antisense sequences to HIV(see above). The results of this experiment are shown in Table 1. TABLE1 % Inhibition of [HIV-1], pg/ml HIV p24 Sample Expt A Expt B Expt AExpt B 2.2.78 pool control 959 ± 49 — 1.9.16 pool 780 error 18.7 —2.10.16 pool 514 554 46.4 42.2

[0479] Both of the pooled clone samples showed inhibition of productionof p24 when compared to the control clones. In the instance of thepooled clone 2.10.16, the degree of inhibition when compared to thecontrol was close to 50%. This pooled clone population of cells wasexamined further as described below.

[0480] At 18 days after infection, the p24 concentration in the growthmedium was determined as described above. The results of thisdetermination are reported in Table 2. TABLE 2 Sample [HIV-1], pg/ml %Inhibition of HIV p24 U937 control 200 — 2.2.78 pool control 220 ± 2  0  2.10.16 pool  12 ± 0.4 94.5

[0481] This table shows that there is approximately 95% inhibition ofp24 antigen production in the pooled clone population of cells whencompared with either the control pooled clone population or the parentcell line.

[0482] On day 24 after viral inoculation, when the cells were assayed bytrypan blue dye exclusion the control pooled clone population were 17%viable, and contained numerous syncytia (multinucleated giant cellscharacteristic of HIV infection). The pooled clone population labeled as2.10.16 were 40-60% viable and had no visible syncytia.

[0483] After day 24, the cells of the control pooled clone culture andthe pooled clone culture were subjected to ficol gradient separation(Pharmacia). This procedure separates the live cells from the dead cellsevery 3-4 days as a routine maintenance procedure. At 35 days, therewere no cells left in the control pooled clone population of cells,while the pooled clone population had viable cells. When these viablecells from the pooled clone population were then assayed for thepresence of the p24 antigen, it was found that the culture line named2.10.16 showed no evidence of the presence of p24 antigen in the culturemedium above the background (0.032+/−0.08 OD compared with 0.039 OD). Inthis experiment, the HIV infected cells had a measured amount of p24antigen that was greater than 2 OD. Thus by this time in the selectionprotocol, the degree of inhibition of the virus was greater than 99%.

[0484] d) HIV Challenge Experiment Number 2

[0485] In this experiment, the pooled clone population identified as2.10.16 (from day 31 of the first challenge) as well as the controlpooled clone population and the parent cell line U937 were infectedagain with the BAL strain of HIV at a multiplicity of 0.10 pfu per cellas described above. After infection, the cells were maintained asdescribed above. At day 9 and day 12 after infection the p24 antigen wasdetermined as described above. The results of this determination arereported in Table 3. TABLE 3 HIV-1 [p24], pg/ml Cell Type day 9 day 12U937   3  5.1 ± 0.4 2.10.16.R1 <1 14.3 ± 1.3

[0486] This table shows that at day 12 there is approximately 66%inhibition of p24 antigen production in the pooled clone population ofcells when compared with the parent cell line.

[0487] When these cells were maintained with separation of the live fromthe dead cells using the ficol gradient every 3-4 days as describedabove it was found at day 21 that there was no evidence of p24 antigenin the 2.10.16 cell lines when compared with the parental cell lineinfected with HIV virus. (Here the comparison is of OD units of the2.10.16 pooled clone population of 0.009 the same number as the controlparental line without infection with >2 OD units.)

[0488] e) Further Characterization of the 2.10.1 6 Cell Line after ThreeCycles of Challenge with HIV Virus

[0489] In this experiment, the pooled clone population identified as2.10.16R1 (from day 21 of the second challenge experiment) and theparent cell line U937 were infected again with the BAL strain of HIV asdescribed above. After infection, the cells were maintained as describedabove. On days 14, 27, and 42 after infection, the p24 antigen wasdetermined as described above for the pooled clone population (nowcalled 2.10.16R2) as well as for the parental cell line U937. Theresults of this determination are reported in Table 4. TABLE 4 HIV-1 p24Day 9 Day 14 Day 27 Day 43 Sample OD pg/ml OD pg/ml OD pg/ml OD pg/mlU937 0.527 122 0.165 25 dead 2.10.16R2 0.12 0 0.009 0 0.030 0 0.026 0buffer 0.013

[0490] This table shows that by day 27 the parental cells havedisappeared from the culture medium. This is consistent with theconclusion that the virus infection has led to the destruction of thecells. In the pooled clone cell population 2.10.16R2, the amount of p24antigen detected in these supernatants is below the sensitivity of theassay procedure. Thus on the third challenge of the original pooledclone cell population there is no evidence of virus growth.

[0491] The parental cell line U937 is known to contain the surfaceantigen CD4+. This parent strain and the strain 2.10.16R2, pooled strainafter 3 cycles of selection, were assayed in a flow cytometer for thepresence of the CD4+ antigen by measuring the binding of mouse CD4+antibody (Becton Dickenson) with fluorescinated goat anti mouse (Tago).As can be seen in FIG. 48, CD4+ antigen is present on the surface of theparental strain and the 2.10.16R2-HIV resistant cell strain. This isevidence that the cells have not been selected to be resistant toinfection by HIV virus through the loss of the adsorption protein,specifically the CD4+ antigen. While the evidence of virus growth basedon the production of the gag antigen, p24, demonstrates that the pooledstrain of cells containing the genetic antisense does not permit thegrowth of virus, further evidence that the virus is not present in thiscell population was obtained using the DNA PCR assay for theidentification of the coding region of the gag gene (the region codingfor the p24 antigen) using the standard Cetus primers which detectvirtually all HIV-1, -2 isolates (Applied BioSystems). As can be seenfrom the FIG. 49 representing UV illumination of the EtBr stained DNA,the + control (using DNA provided in the kit) gave a band of theexpected size (lane 1), while several dilution of the amplificationproducts of 2.10.16R2 DNA did not show such a band.

[0492] These data demonstrate that cell lines can be developed usingantisense constructs that maintain their CD4+ phenotype. These celllines do not support the growth of the HIV virus as measured both by theproduction of the p24 antigen and measured with the quick DNA PCR kit ofCetus. In addition these cell strains have been shown to survivemultiple challenge from infectious HIV virus.

Example 31 Testing the Anti HIV U1 Constructs in Cells

[0493] Inhibition of synthesis of beta-galactosidase activity:

[0494] a) Eukaryotic Vector Carrying Target Sequence A Upstream of theBeta-galactosidase Gene

[0495] The A segment (from the tar sequence of HIV) of target DNA wasisolated as described above. This segment was cloned into the Kpn1 BamH1site of the eukaryotic vector pSV Lac Z(invitrogen), that carries Lac Zcoding sequences and SV40 enhancer and promoter and poly A signalsequences. The cloning sites is between these sequences. The cloningsequence is diagrammed in the attached figure (FIG. 50).

[0496] b) Expression of Beta-galactosidase Activity in StablyTransfected U937 Cells

[0497] U937 cells were transformed using the Lipofectin proceduredescribed above. In this experiment positive clones were selected aszeocin resistant. 5 separate transfected cell populations were isolated.These cells were 1. U937 cells untransfected; 2. U937 cells transformedwith the HIV A clone alone; 3. U937 cells transfected with the HIV Aclone and then a second time with the U1 antisense A clone (see abovefor the description of the clone the second transfection was selected asG418 resistant); 4. U937 cells cotransfected with the HIV A clone andthe U1 antisense ABC clone (again see above for a description of theclone); and 5. U937 cells cotransfected with the HIV A clone and theU1-null DNA clone (again see above for a description of the clone).

[0498] Log phase cells of U937 (both stably transfected anduntransfected) were washed free of medium with 1×PBS containing 10 mMMg⁺⁺ and 1 mM Ca⁺⁺. The washed cells were fixed lightly (5 minutes) atroom temperature in PBS containing 2% formaldehyde and 0.05%glutaraldehyde. The fixative was removed and the cells were washed freeof fixative with two washes with PBS. The washed fixed cells were thensuspended in staining solution (PBS containing 5 mM potassiumferrocyanide and 2 mM MgCl₂) containing 1 mg/ml X-gal (BRL) andincubated at 37° C. for 2 hours to overnight. The cells were examinedunder a microscope at 40x.

[0499] The results of this experiment are illustrated in FIG. 51 (lowerset of data). The positive production of the enzyme beta-galactosidaseis assayed by the production of a blue precipitate in the cytoplasm ofthe transfected cells. No blue is detected in cell lines 1, 3 and 4while blue spots are detected in the cytoplasm of the cell line 2 and 5.These data demonstrate that the production of the enzymebeta-galactosidase that is shown as a blue stain in cell line 2 with theHIV A clone alone or in cell line 5 where both the HIV A clone and thenull DNA control is not seen when either the antisense U1 A clone iscotransfected with the HIV A clone (cell line 3) or the antisense U1 ABCclone is cotransfected with the HIV A clone (cell line 4). Thus thepresence of the antisense A sequence in the cell lines with this HIV Aclone expressing the enzyme beta-galactosidase blocks the production ofthis enzyme.

[0500] c. Expression of Beta-galactosidase Activity in Extracts:

[0501] To measure enzyme activity by soluble assay (FIG. 51, upper setof data) extracts were prepared from loge-phase cultures either bysonication or repeated freeze-thawing. The log-phase cells (5×10⁶ cellsper ml) were washed free of medium with PBS containing 10 mM Mg⁺⁺ and 1mM Ca⁺⁺. The washed cells were suspended in 250 mM Tris-Cl, pH 7.5 andfreeze-thawed 3 times or alternatively sonicated 5 minutes at maximumoutput. The crude Iysate was centrifuged and enzyme activity wasmeasured in clear supernatants by hydrolysis of the lactose analog ONPG(Sigma). When this substrate is cleaved by the enzyme to make ONP ayellow colored compound produced. Thus the beta-alactosidase activitycan be monitored by observing the change in absorbance at 420 nm.Extracts prepared from cells that are stably transfected with the HIV Aclone produce a yellow color in 30 minutes at 37° C., whereas theextracts prepared from untransfected cells remain colorless even afterincubation over night.

[0502] The 5 transfected cell lines were assayed using this solubleassay format and the results are reported in table 5. From this table itcan be seen that the U1 anti-A transfected cells do not have measurableamounts of beta-galactosidase activity. (Compare line 3 with lines 2 and5.) Also it can be seen that the U1 anti-ABC clones do not showmeasurable amounts of beta-galactosidase activity. (Compare line 4 withlines 2 and 5.) These results confirm the results from the in situ assayof the effect of the U1 anti A and anti ABC clones on the production ofbeta-galactosidase activity of clones that have the A target cloned intotheir sequences.

Example 32

[0503] Asymptomatic HIV positive patients are given pre-treatmentevaluations including medical histories; physical examinations, bloodchemistries including CBCs, differential counts, platelet counts; bloodchemistries including glucose, calcium, protein, albumin, uric acid,phosphate; Blood Urea Nitrogen and creatinine; Urinalysis;electrocardiogram and chest X-ray; p24 antigen level; CD4 counts; PCR todetermine viral load. The p24 antigen, CD4 counts and PCR are done atweekly intervals for 4 weeks prior to removal of cells in order toestablish baseline data, and these assays are continued biweeklythroughout the period of treatment. Blood is removed from patients andthe peripheral blood mononuclear cells are separated from erythrocytesand neutrophils by Ficoll-Hypaque centrifucation. After washing, thePBMCs are depleted of CD8+ cells by the use of murine anti-humanCD8-coated flasks D2 CELLector™ Flasks, Applied Immune Sciences). Cellswhich do not adhere to the surface of the flasks are cells assayed forcellular phenotype by flow cytometry and then activated with OKT3antibody in serum-free medium.

[0504] The OKT3-activated cells are resuspended at a concentration of1-2×10⁵ cells/ml in fresh medium containing 60 units/ml of IL-2. Thecells are expanded to about 2×10⁶/ml.

[0505] A retrovirus vector containing sequences for the expression ofantisense RNA directed at HIV is grown in a packaging cell line. A DNAconstruct (described in Example F1 is introduced into retrovirus vectorLNL6, which contains a neomycin resistance marker. The cells aretransduced by resuspension in culture medium to a concentration ofapproximately 10⁵ cells/ml and mixing with culture supernatant from theretrovirus vector infected cells to provide an MOI of approximately 1.0.Five mg/ml protamine sulfate are added and the mixture is incubated at37° C. for 6 hours. The cells are washed three times and placed in G418containing culture medium. This transduction procedure is repeated dailyfor three consecutive days.

[0506] After 7 days in G418 selection medium the G418 is removed and thecells are expanded in the presence of growth factors (as describedabove). When sufficient cells are produced, they are harvested, washedand resuspended in physiological saline for infusion into the patient.Cellular phenotype is measured by flow cytometry measurements.

[0507] This antisense treatment is supplemented by treatment withsoluble CD4 protein. Administration commences immediately after theadministration of HIV therapy according to the method of (Husson et al.,1992).

[0508] This supplemented gene therapy is further supplemented byconcurrent administration of AZT.

[0509] Many obvious variations might be suggested to those of ordinaryskill in the art in light of the above detailed description of theinvention. All such variations are fully embraced by the scope andspirit of the present invention as set forth in the claims which nowfollow.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:42 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:20 amino acids (B) TYPE:amino acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (ix)SEQUENCE DESCRIPTION:SEQ ID NO:1: Gly Phe Phe Gly Ala Ile Ala Gly PheLeu Glu Gly Gly Trp Glu Gly 1 5 10 15 Met Ile Ala Gly 20 (2) INFORMATIONFOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs(B) TYPE:nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) SEQUENCEDESCRIPTION:SEQ ID NO:2: TGCTCTCTAA GGGTCTACTC 20 (2) INFORMATION FORSEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:15 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) SEQUENCEDESCRIPTION:SEQ ID NO:3: CTCTAAGGTA AATAT 15 (2) INFORMATION FOR SEQ IDNO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) SEQUENCEDESCRIPTION:SEQ ID NO:4: TGTATTTTAG ATTCAA 16 (2) INFORMATION FOR SEQ IDNO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:5: TGCTCTCTAA GGTAAATAT 19 (2) INFORMATION FOR SEQID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:6: TGTATTTTAG GGTCTACTC 19 (2) INFORMATION FOR SEQID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:7: UGCUCUCUAA GGUAAAUAU 19 (2) INFORMATION FOR SEQID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:8: UGUAUUUUAG GGUCUACUC 19 (2) INFORMATION FOR SEQID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:9: UGCUCUCUAA GGGUCUACUC 20 (2) INFORMATION FORSEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:49 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:10: GGAATTCGTC TCGAGCTCTG ATCACCACCA TGGACACGAT TAACATCGC 49 (2)INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:55 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:11: GACTAGTTGG TCTCGTCTCT TTTTTGGAGG AGTGTCGTTCTTAGCGATGT TAATC 55 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH:46 base pairs (B) TYPE:nucleic acid (C)STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:12: GGAATTCGTCTCGGAGAAAG GTAAAATTCT CTGACATCGA ACTGGC 46 (2) INFORMATION FOR SEQ IDNO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:33 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:13: GACTAGTGGT CTCCCCTTAG AGAGCATGTC AGC 33 (2) INFORMATION FORSEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:33 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:14: GGAATTCGGT CTCGGGTCTA CTCGGTGGCG AGG 33 (2) INFORMATION FORSEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:27 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:15: GACTAGTCGT TACGCGAACG CAAAGTC 27 (2) INFORMATION FOR SEQ IDNO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:36 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:16: GGAATTCGTC TCTAAGGTAA ATATAAAATT TTTAAG 36 (2) INFORMATION FORSEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:40 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:17: GACTAGTCGT CTCTGACCCT AAAATACACA AACAATTAGA 40 (2) INFORMATIONFOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:92 base pairs(B) TYPE:nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:18: GGAATTCGTC TCGAGCTCTG ATCACCACCA TGGACACGATTAACATCGCT AAGAACGACA 60 CTCCTCCAAA AAAGAGACGA GACCAACTAG TC 92 (2)INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:46 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:double (D)TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:19: GGAATTCGTC TCGGAGAAAGGTAAAATTCT CTGACATCGA ACTGGC 46 (2) INFORMATION FOR SEQ ID NO:20: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH:106 base pairs (B) TYPE:nucleicacid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ IDNO:20: GGAATTCGTC TCGAGCTCTG ATCACCACCA TGGACACGAT TAACATCGCT AAGAACGACA60 CTCCTCCAAA AAAGAGAAAG GTAAAATTCT CTGACATCGA ACTGGC 106 (2)INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:50 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:double (D)TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO(ix) SEQUENCE DESCRIPTION:SEQ ID NO:21: ATGGACACGA TTAACATCGC TAAGAACGACTTCTCTGACA TCGAACTGGC 50 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH:77 base pairs (B) TYPE:nucleic acid (C)STRANDEDNESS:double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ IDNO:22: ATGGACACGA TTAACATCGC TAAGAACGAC ACTCCTCCAA AAAAGAGAAA GGTAAAATTC60 TCTGACATCG AACTGGC 77 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCECHARACTERISTICS: (A) LENGTH:69 base pairs (B) TYPE:nucleic acid (C)STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:23: GATCATTAGACCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA 60 AGCCTCAAG 69(2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:69 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:24: GATCCTTGAG GCTTAAGCAG TGGGTTCCCT AGTTAGCCAGAGAGCTCCCA GGCTCAGATC 60 TGGTCTAAT 69 (2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH:61 base pairs (B) TYPE:nucleicacid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE:other nucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:25: GATCACCTTAGGCTCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 60 G 61 (2)INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:61 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:26: GATCCTTGAG GAGGTCTTCG TCGCTGTCTC CGCTTCTTCCTGCCATAGGA GAGCCTAAGG 60 T 61 (2) INFORMATION FOR SEQ ID NO:27: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH:62 base pairs (B) TYPE:nucleic acid(C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:27: GATCATAGTGAATAGAGTTA GGCAGGGATA CTCACCATTA TCGTTTCAGA CCCACCTCCC 60 AG 62 (2)INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:62 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:28: GATCCTGGGA GGTGGGTCTG AAACGATAAT GGTGAGTATCCCTGCCTAAC TCTATTCACT 60 AT 62 (2) INFORMATION FOR SEQ ID NO:29: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH:30 base pairs (B) TYPE:nucleic acid(C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (iv) ORIGINAL SOURCE: (A) ORGANISM: TER-1 (x) SEQUENCEDESCRIPTION:SEQ ID NO:29: AATCTAGAGC TAACAAAGCC CGAAAGGAAG 30 (2)INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:28 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:30: TTCTGCAGAT ATAGTTCCTC CTTTCAGC 28 (2)INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:70 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:31: TCGAGCCATG GCTTAAGGAT CCGTACGTCC GGAGCTAGCGGGCCCATCGA TACTAGTTAA 60 ATGCAGATCT 70 (2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH:70 base pairs (B) TYPE:nucleicacid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE:other nucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:32: CTAGAGATCTGCATTTAACT AGTATCGATG GGCCCGCTAG CTCCGGACGT ACGGATCCTT 60 AAGCCATGGC 70(2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:29 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:double (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:33: CATGAAATTA ATTCGACTCA CTATACGGA 29 (2)INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:29 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:double (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:34: TTTAATTAAG CTGAGTGATA TGCCTCTAG 29 (2)INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:72 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:35: GATCCGGATT GAGGCTTAAG CAGTGGGTTC CCTAGTTAGCCAGAGAGCTC CCAGGCTCAG 60 ATCTGGTCTA AT 72 (2) INFORMATION FOR SEQ IDNO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:72 base pairs (B)TYPE:nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii)MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc =“oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQID NO:36: CCGGATTAGA CCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAACCCACTGCTTA 60 AGCCTCAATC CG 72 (2) INFORMATION FOR SEQ ID NO:37: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH:66 base pairs (B) TYPE:nucleic acid(C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:37: GATCCGGACCTTGAGGAGGT CTTCGTCGCT GTCTCCGCTT CTTCCTGCCA TAGGAGAGCC 60 TAAGGT 66 (2)INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:66 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:38: CCGGACCTTA GGCTCTCCTA TGGCAGGAAG AAGCGGAGACAGCGACGAAG ACCTCCTCAA 60 GGTCCG 66 (2) INFORMATION FOR SEQ ID NO:39: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH:65 base pairs (B) TYPE:nucleic acid(C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:39: GATCCGGATGGGAGGTGGGT CTGAAACGAT AATGGTGAGT ATCCCTGCCT AACTCTATTC 60 ACTAT 65 (2)INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:65 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:40: CCGGATAGTG AATAGAGTTA GGCAGGGATA CTCACCATTATCGTTTCAGA CCCACCTCCC 60 ATCCG 65 (2) INFORMATION FOR SEQ ID NO:41: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH:67 base pairs (B) TYPE:nucleic acid(C) STRANDEDNESS:single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: othernucleic acid (A) DESCRIPTION: /desc = “oligonucleotide” (iii)HYPOTHETICAL: YES (ix) SEQUENCE DESCRIPTION:SEQ ID NO:41: GATCAGCATGCCTGCAGGTC GACTCTAGAC CCGGGTACCG AGCTCGCCCT ATAGTGAGTC 60 GTATTAT 67 (2)INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH:67 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS:single (D)TOPOLOGY:linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION:/desc = “oligonucleotide” (iii) HYPOTHETICAL: YES (ix) SEQUENCEDESCRIPTION:SEQ ID NO:42: CCGGATAATA CGACTCACTA TAGGGCGAGC TCGGTACCCGGGTCTAGAGT CGACCTGCAG 60 GCATGCT 67

What is claimed is:
 1. A non-naturally occurring construct which whenpresent in a cell produces a product, said construct comprising at leastone modified nucleotide, a nucleotide analog or a non-nucleic acidentity, or a combination of the foregoing.
 2. The construct of claim 1,wherein said construct or a portion thereof is linear, circular orbranched.
 3. The construct of claim 1, wherein said construct or aportion thereof is single-stranded, double-stranded, partiallydouble-stranded or triple-stranded.
 4. The construct of claim 3, havingat least one terminus, said terminus comprising a polynucleotide tail.5. The construct of claim 4, wherein said polynucleotide tail ishybridized to a complementary polynucleotide sequence.
 6. The constructof claim 1, wherein said construct comprises DNA, RNA, a DNA-RNA hybrid,a DNA-RNA chimera, or a combination of the foregoing.
 7. The constructof claim 1, wherein said modified nucleotide has been chemicallymodified.
 8. The construct of claim 6, wherein said chemicalmodification has been effected to a moiety in independently selectedfrom a base, a sugar, and a phosphate, or a combination thereof.
 9. Theconstruct of claim 1, wherein at least one of said nucleotide analog oranalogs have been modified on the backbone or sidechain or both.
 10. Theconstruct of claim 1, wherein said non-nucleic acid entity is attachedto a single strand or to both strands of said sequence segment.
 11. Theconstruct of claim 1, wherein said non-nucleic acid entity or entitiesare selected from a natural or synthetic polymer, and a natural orsynthetic ligand, or a combination thereof.
 12. The construct of claim11, wherein said natural polymer comprises a modified or unmodifiedmember selected from a polypeptide, a protein, a polysaccharide, a fattyacid, and a fatty acid ester, or a combination of the foregoing.
 13. Theconstruct of claim 11, wherein said synthetic polymer comprises ahomopolymer or heteropolymer.
 14. The construct of claim 13, whereinsaid homopolymer or heteropolymer carries a net negative charge or a netpositive charge.
 15. The construct of claim 1, wherein said constructexhibits a further biological activity imparted by said modifiednucleotide, said nucleotide analog, said nucleic acid entity, a ligand,or a combination of the foregoing.
 16. The construct of claim 15,wherein said biological activity is selected from nuclease resistance,cell recognition, cell binding, and cellular or nuclear localization, ora combination of the foregoing.
 17. The construct of claim 1, furthercomprising at least one ligand attached covalently or noncovalently tosaid modified nucleotide or nucleotides, nucleotide analog or analogs,non-nucleic acid entity or entities, or a combination thereof.
 18. Theconstruct of claim 17, wherein said ligand or ligands are attached to asingle stranded segment, a double stranded segment, a single strandedconstruct tail, or a sequence complementary to a construct tail, or acombination of the foregoing.
 19. The construct of claim 17, whereinsaid ligand or ligands are selected from macromolecules and smallmolecules, or a combination of both.
 20. The construct of claim 1,wherein said construct carries a net positive charge, or a net negativecharge, or is neutral or hydrophobic.
 21. The construct of claim 1,wherein said construct comprises unmodified nucleotides and at least onemember selected from one or more nucleotide analogs and non-nucleic acidentities, or a combination thereof.
 22. A construct which when presentin a cell produces a product, said construct being bound non-ionicallyto an entity comprising a chemical modification or a ligand.
 23. Theconstruct of claim 22, having at least one terminus, said terminuscomprising a polynucleotide tail.
 24. The construct of claim 23, whereinsaid polynucleotide tail is hybridized to a complementary polynucleotidesequence.
 25. The construct of claim 24, wherein an antibody is bound tosaid hybridized polynucleotide tail sequences.
 26. The construct ofclaim 25, wherein said antibody comprises a polyclonal or monoclonalantibody.
 27. A composition comprising: (a) a non-natural entity whichcomprises: at least one domain to a nucleic acid component; and at leastone domain to a cell of interest; and (b) said nucleic acid component;wherein the domain or domains to said nucleic acid component aredifferent from the domain or domains to said cell.
 28. The compositionof claim 27, wherein said entity comprises a binder.
 29. The compositionof claim 28, wherein said binder and said domain are the same.
 30. Thecomposition of claim 28, wherein said binder and said domain aredifferent.
 31. The composition of claim 28, wherein said binder isselected from a polymer, a matrix, a support, or a combination of any ofthe foregoing.
 32. The composition of claim 27, wherein said nucleicacid component is selected from a nucleic acid, a nucleic acidconstruct, a nucleic acid conjugate, a virus, a viral fragment, a viralvector, a viroid, a phage, a plasmid, a plasmid vector, a bacterium anda bacterial fragment, or a combination of the foregoing.
 33. Thecomposition of claim 27, wherein said cell is prokaryotic or eukaryotic.34. The composition of claim 27, wherein said domains are attachedcovalently or noncovalently, or through a binder, or a combinationthereof.
 35. The composition of claim 34, wherein said noncovalentbinding is selected from ionic interactions and hydrophobicinteractions, or a combination thereof.
 36. The composition of claim 35,wherein said noncovalent binding comprises a specific complex.
 37. Thecomposition of claim 36, wherein said specific complex is mediated by aligand binding receptor.
 38. The composition of claim 37, wherein saidligand binding receptor is selected from a polynucleotide sequence to berecognized by its complementary sequence, an antigen to be recognized byits corresponding monoclonal or polyclonal antibody, an antibody to berecognized by its corresponding antigen, a lectin to be recongnized byits corresponding sugar, a hormone to be recognized by its receptor, areceptor to be recognized by its hormone, an inhibitor to be recognizedby its enzyme, an enzyme to be recognized by its inhibitor, a cofactorto be recognized by its cofactor enzyme binding site, a cofactor enzymebinding site to be recognized by its cofactor, a binding ligand to berecognized by its substrate, or a combination of the foregoing.
 39. Thecomposition of claim 28, wherein the domain to said nucleic acidcomponent and the domain to said cell of interest are natural, and saidbinder is attached to said nucleic acid component by means other than anatural binding site.
 40. The composition of claim 39, wherein saidbinder comprises modified fibronectin or modified polylysine, or both.41. The composition of claim 27, wherein said cell of interest iscontained within an organism.
 42. The composition of claim 27, furthercomprising said cell of interest.
 43. A method of introducing a nucleicacid component into a cell comprising: (a) providing the composition ofclaim 27; and (b) administering said composition.
 44. The method ofclaim 43, wherein administering is carried out in vivo.
 45. The methodof claim 43, wherein administering is carried out ex vivo.
 46. A kit forintroducing a nucleic acid component into a cell of interest, comprisingin packaged containers or combination: (a) a non-natural entity whichcomprises at least one domain to said nucleic acid component, and adomain to said cell of interest; (b) a nucleic acid component,optionally with (c) buffers and instructions.
 47. A compositioncomprising: an entity which comprises at least one domain to a cell ofinterest, wherein said domain or domains are attached to a nucleic acidcomponent which is in non-double stranded form.
 48. The composition ofclaim 47, wherein said entity comprises a binder.
 49. The composition ofclaim 48, wherein said binder and said domain are the same.
 50. Thecomposition of claim 48, wherein said binder and said domain aredifferent.
 51. The composition of claim 48, wherein said binder isselected from a polymer, a matrix, a support, or a combination of any ofthe foregoing.
 52. The composition of claim 47, wherein said cell isprokaryotic or eukaryotic.
 53. The composition of claim 47, wherein saidnucleic acid component is selected from a nucleic acid, a nucleic acidconstruct, a nucleic acid conjugate, a virus, a viral fragment, a viralvector, a viroid, a phage, a plasmid, a plasmid vector, a bacterium anda bacterial fragment, or a combination of the foregoing.
 54. Thecomposition of claim 47, wherein said domain is selected from covalentbonding and noncovalent binding, or a combination thereof.
 55. Thecomposition of claim 54, wherein said noncovalent binding is selectedfrom ionic interactions and hydrophobic interactions, or a combinationthereof.
 56. The composition of claim 54, wherein said noncovalentbinding comprises a specific complex.
 57. The composition of claim 56,wherein said specific complex is mediated by a ligand binding receptor.58. The composition of claim 57, wherein said ligand binding receptor isselected from a polynucleotide sequence to be recognized by itscomplementary sequence, an antigen to be recognized by its correspondingmonoclonal or polyclonal antibody, an antibody to be recognized by itscorresponding antigen, a lectin to be recognized by its correspondingsugar, a hormone to be recognized by its receptor, a receptor to berecognized by its hormone, an inhibitor to be recognized by its enzyme,an enzyme to be recognized by its inhibitor, a cofactor to be recognizedby its cofactor enzyme binding site, a cofactor enzyme binding site tobe recognized by its cofactor, a binding ligand to be recognized by itssubstrate, or a combination of the foregoing.
 59. The composition ofclaim 47, wherein said cell of interest is contained within an organism.60. The composition of claim 47, further comprising said cell ofinterest.
 61. A method of introducing a nucleic acid component into acell comprising: (a) providing the composition of claim 47; and (b)administering said composition.
 62. The method of claim 61, whereinadministering is carried out in vivo.
 63. The method of claim 61,wherein administering is carried out ex vivo.
 64. A kit for introducinga nucleic acid component into a cell of interest, comprising in packagedcontainers or combinations: (a) an entity which comprises a domain tosaid cell of interest, wherein said domain is attached to a nucleic acidcomponent which is in non-double stranded form, optionally with (b)buffers and instructions.
 65. A composition comprising: an entity whichcomprises a domain to a nucleic acid component, wherein said domain isattached to a cell of interest.
 66. The composition of claim 65, whereinsaid entity comprises a binder.
 67. The composition of claim 66, whereinsaid binder and said domain are the same.
 68. The composition of claim66, wherein said binder and said domain are different.
 69. Thecomposition of claim 66, wherein said binder is selected from a polymer,a matrix, a support, or a combination of any of the foregoing.
 70. Thecomposition of claim 66, wherein said nucleic acid component is selectedfrom a nucleic acid, a nucleic acid construct, a nucleic acid conjugate,a virus, a viral fragment, a viral vector, a viroid, a phage, a plasmid,a plasmid vector, a bacterium and a bacterial fragment, or a combinationof the foregoing.
 71. The composition of claim 65, wherein said cell iseukaryotic or prokaryotic.
 72. The composition of claim 65, wherein saiddomain is selected from covalent bonding and noncovalent binding, or acombination thereof.
 73. The composition of claim 72, wherein saidnoncovalent binding is selected from ionic interactions and hydrophobicinteractions, or a combination thereof.
 74. The composition of claim 72,wherein said noncovalent binding comprises a specific complex.
 75. Thecomposition of claim 74, wherein said specific complex is mediated by aligand binding receptor.
 76. The composition of claim 75, wherein saidligand binding receptor is selected from a polynucleotide sequence to berecognized by its complementary sequence, an antigen to be recognized byits corresponding monoclonal or polyclonal antibody, an antibody to berecognized by its corresponding antigen, a lectin to be recognized byits corresponding sugar, a hormone to be recognized by its receptor, areceptor to be recognized by its hormone, an inhibitor to be recognizedby its enzyme, an enzyme to be recognized by its inhibitor, a cofactorto be recognized by its cofactor enzyme binding site, a cofactor enzymebinding site to be recognized by its cofactor, a binding ligand to berecognized by its substrate, or a combination of the foregoing.
 77. Thecomposition of claim 65, further comprising said cell of interest. 78.The composition of claim 65, wherein said cell of interest is containedwithin an organism.
 79. A method of introducing a nucleic acid componentinto a cell comprising: (a) providing the composition of claim 65; and(b) administering said composition.
 80. The method of claim 79, whereinadministering is carried out in vivo.
 81. The method of claim 79,wherein administering is carried out ex vivo.
 82. A kit for introducinga nucleic acid component into a cell of interest, comprising in packagedcontainers or combination: (a) an entity which comprises a domain tosaid nucleic acid component, wherein said domain is attached to saidcell of interest, optionally with (b) buffers and instructions.
 83. Amultimeric complex composition comprising more than one monomeric unitattached. (a) to each other through polymeric interactions, or (b) to abinding matrix through polymeric interactions, or (c) both (a) and (b)84. The composition of claim 83, wherein the polymer or oligomer of saidmonomeric unit is linear or branched.
 85. The composition of claim 83,wherein the polymer or oligomer of said monomeric unit comprises ofhomopolymer or heteropolymer.
 86. The composition of claim 83, whereinsaid monomeric unit comprises an analyte-specific moiety.
 87. Thecomposition of claim 86, wherein said analyte-specific moiety is capableof recognizing a component in a biological system.
 88. The compositionof claim 87, wherein said biological system is selected from a virus, aphage, a bacterium, a cell or cellular material, a tissue, an organ andan organism, or a combination thereof.
 89. The composition of claim 83,wherein said monomeric unit is selected from a naturally occurringcompound, a modified natural compound, a synthetic compound and arecombinantly produced compound, or a combination thereof.
 90. Thecomposition of claim 83, wherein said analyte-specific moiety is derivedor selected from a protein, a polysaccharide, a fatty acid or fatty acidester and a polynucleotide, or a combination of the foregoing.
 91. Thecomposition of claim 90, wherein said protein is selected from anantibody, a hormone, a growth factor, a lymphokine or cytokine and acellular matrix protein, or a combination of any of the foregoing. 92.The composition of claim 91, wherein said antibody comprises apolyclonal or monoclonal antibody.
 93. The composition of claim 90,wherein said polynucleotide is linear or circular.
 94. The compositionof claim 90, wherein said polynucleotide is single stranded.
 95. Thecomposition of claim 83, wherein the polymer or oligomer of said bindingmatrix is linear or branched.
 96. The composition of claim 83, whereinthe polymer or oligomer of said binding matrix comprises a homopolymeror heteropolymer.
 97. The composition of claim 83, wherein said bindingmatrix is selected from a naturally occurring compound, a modifiednatural compound, a synthetic compound and a recombinantly producedcompound, or a combination thereof.
 98. The composition of claim 83,wherein said binding matrix comprises a member selected from apolypeptide, a polynucleotide and a polysaccharide, or a combinationthereof.
 99. The composition of claim 83, wherein said polymericinteractions are selected from ionic interactions, hydrogen bonding,dipole-dipole interactions, or a combination of the foregoing.
 100. Thecomposition of claim 99, wherein said ionic interactions comprisepolycationic interactions or polycationic interactions.
 101. Thecomposition of claim 83, further comprising an entity attached to saidbinding matrix.
 102. The composition of claim 101, wherein said entitycomprises a ligand or a compound which increases binding of the bindingmatrix.
 103. The composition of claim 83, in homogeneous form.
 104. Thecomposition of claim 83, in heterogeneous form.
 105. A process fordelivering a cell effector to a cell, comprising: providing themultimeric complex composition of claim 83 wherein said monomeric unitcomprises said cell effector; and administering said composition. 106.The process of claim 105, wherein said composition is delivered in vivo.107. The process of claim 105, wherein said composition is delivered exvivo.
 108. The process of claim 105, wherein said cell is contained inan organism.
 109. A process for delivering a gene or fragment thereof toa cell, comprising: providing the multimeric complex composition ofclaim 83, wherein said monomeric unit comprises said gene or genefragment; and administering said composition.
 110. The process of claim109, wherein said composition is delivered in vivo.
 111. The process ofclaim 109, wherein said composition is delivered ex vivo.
 112. Theprocess of claim 109, wherein said cell is contained in a organism. 113.A multimeric composition comprising more than one component attached toa charged polymer, wherein said charged polymer is selected from apolycationic polymer, a polyionic polymer, a polynucleotide, a modifiedpolynucleotide and a polynucleotide analog, or a combination of theforegoing.
 114. The multimeric composition of claim 113, wherein saidcomponent comprises a protein.
 115. The multimeric composition of claim114, wherein said protein is selected from an antibody and an F(ab′)₂fragment, or both.
 116. The multimeric composition of claim 115, whereinsaid antibody comprises a polyclonal or monoclonal antibody.
 117. Themultimeric composition of claim 115, wherein said antibody is furthercomplex with a target comprising an enzyme.
 118. A nucleic acidconstruct which when introduced into a cell codes for and expresses anon-native polymerase, said polymerase being capable of producing morethan one copy of a nucleic acid sequence from said construct.
 119. Theconstruct of claim 118, further comprising a recognition site for saidnon-native polymerase.
 120. The construct of claim 119, wherein saidrecognition site is complementary to a primer for said non-nativepolymerase.
 121. The construct of claim 120, wherein said primercomprises transfer RNA (tRNA).
 122. The construct of claim 118, whereinsaid non-native polymerase comprises a member selected from DNApolymerase, RNA polymerase and reverse transcriptase, or a combinationthereof.
 123. The construct of claim 122, wherein said RNA polymerasecomprises a bacteriophage RNA polymerase.
 124. The construct of claim123, wherein said bacteriophage RNA polymerase is selected from T3, T7and SP6, or a combination thereof.
 125. The construct of claim 122,further comprising a promoter for said RNA polymerase.
 126. Theconstruct of claim 118, wherein said nucleic acid produced from saidconstruct is selected from DNA, RNA, a DNA-RNA hybrid and a DNA-RNAchimera, or a combination of the foregoing.
 127. The construct of claim126, wherein said DNA or RNA comprises sense or antisense, or both. 128.A nucleic acid construct which when introduced into a cell produces anucleic acid product comprising a non-native processing element, whichwhen in a compatible cell, said processing element is substantiallyremoved during processing.
 129. The construct of claim 128, wherein saidprocessing element comprises an RNA processing element.
 130. Theconstruct of claim 129, wherein said RNA processing element is selectedfrom an intron, a polyadenylation signal and a capping element, or acombination of the foregoing.
 131. The construct of claim 128, whereinsaid nucleic acid product is single stranded.
 132. The construct ofclaim 128, wherein said nucleic acid product is selected from antisenseRNA, antisense DNA, sense RNA, sense DNA, a ribozyme and a proteinbinding nucleic acid sequence, or a combination of the foregoing. 133.The composition of claim 132, wherein said protein binding nucleic acidsequence comprises a decoy that binds a protein required for viralassembly or viral replication.
 134. A process for selectively expressinga nucleic acid product in a cell, which product requires processing forfunctioning, said process comprising: (i) providing a nucleic acidconstruct which when introduced into a cell produces a nucleic acidproduct comprising a non-native processing element, which when in acompatible cell, said processing element is substantially removed duringprocessing; and (ii) introducing said construct into said cell.
 135. Theprocess of claim 134, wherein said processing element comprises an RNAprocessing element selected from an intron, a polyadenylation signal anda capping element, or a combination of the foregoing.
 136. The processof claim 134, wherein said nucleic acid product is selected fromantisense RNA, antisense DNA, sense RNA, sense DNA, a ribozyme and aprotein binding nucleic acid sequence, or a combination of theforegoing.
 137. The process of claim 134, wherein said construct isintroduced ex vivo into said cell.
 138. The process of claim 137,wherein said construct is introduced in vivo into said cell.
 139. Theprocess of claim 134, wherein said construct is introduced into abiological system containing said cell.
 140. The process of claim 139,wherein said biological system is selected from an organism, an organ, atissue and a culture, or a combination of the foregoing.
 141. Acomposition comprising a primary nucleic acid component which uponintroduction into a cell produces a secondary nucleic acid componentwhich is capable of producing a nucleic acid product, or a tertiarynucleic acid component, or both, wherein said primary nucleic acidcomponent is not obtained with said secondary or tertiary component orsaid nucleic acid product.
 142. The composition of claim 141, whereinsaid cell is eukaroytic or prokaryotic.
 143. The composition of claim141, wherein said primary nucleic acid component is selected from anucleic acid, a nucleic acid construct, a nucleic acid conjugate, avirus, a viral fragment, a viral vector, a viroid, a phage, a phage, aplasmid, a plasmid vector, a bacterium and a bacterial fragment, or acombination of the foregoing.
 144. The composition of claim 141, whereinsaid primary nucleic acid component is single-stranded, double-strandedor partially double-stranded.
 145. The composition of claim 141, whereinsaid primary nucleic acid component is selected from DNA, RNA andnucleic acid analogs, or a combination thereof.
 146. The composition ofclaim 145, wherein said DNA, RNA or both are modified.
 147. Thecomposition of claim 141, wherein said secondary nucleic acid componentor said tertiary nucleic acid component is selected from DNA, RNA, aDNA-RNA hybrid and a DNA-RNA chimera, or a combination of the foregoing.148. The composition of claim 141, further comprising a signalprocessing sequence.
 149. The composition of claim 148, wherein saidsignal processing sequence is selected from a promoter, an initiator, aterminator, an intron and a cellular localization element, or acombination of the foregoing.
 150. The composition of claim 148, whereinsaid signal processing sequence is contained in an element selected fromsaid primary nucleic acid component, said secondary nucleic acidcomponent, said nucleic acid product and said tertiary nucleic acidcomponent, or a combination of the foregoing.
 151. The composition ofclaim 141, wherein said nucleic acid product is single-stranded. 152.The composition of claim 141, said nucleic acid product is selected fromantisense RNA, antisense DNA, a ribozyme and a protein binding nucleicacid sequence, or a combination of the foregoing.
 153. The compositionof claim 152, wherein said protein binding nucleic acid sequencecomprises a decoy that binds a protein required for viral assembly orviral replication.
 154. The composition of claim 141, wherein saidcomponent or nucleic acid production is mediated by a vector.
 155. Thecomposition of claim 154, wherein said vector is selected from a viralvector, a phage vector and a plasmid vector, or a combination thereof.156. A cell containing the composition of claim
 141. 157. The cell ofclaim 156, wherein said cell is a eukaryotic or prokaryotic.
 158. Thecell of claim 156, wherein said composition has been introduced ex vivointo said cell.
 159. The cell of claim 156, wherein said composition hasbeen introduced in vivo into said cell.
 160. A secondary or tertiarynucleic acid component or nucleic acid product produced from thecomposition of claim
 1. 161. A composition of matter comprising anucleic acid component which when present in a cell produces anon-natural nucleic acid product, which product composes (i) a portionof a localizing localizing entity, and (ii) a nucleic acid sequence ofinterest.
 162. The composition of claim 161, wherein said portion of thelocalizing entity (i) is sufficient to permit localization of saidnon-natural nucleic acid product.
 163. The composition of claim 161,wherein said said portion of the localizing entity (i) comprises acytoplasmic or nuclear localization signalling sequence.
 164. Thecomposition of claim 161, wherein said nucleic acid sequence of interest(ii) is selected from DNA, RNA, a DNA-RNA hybrid and a DNA-RNA chimera,or a combination of the foregoing.
 165. The composition of claim 164,wherein said RNA comprises a nuclear localized RNA complexed withprotein molecules.
 166. The composition of claim 165, wherein saidnuclear localized RNA comprises a snRNA.
 167. The composition of claim166, wherein said snRNA comprises U1 or U2, or both.
 168. Thecomposition of claim 161, wherein said non-natural nucleic acid productis single-stranded.
 169. The composition of claim 161, wherein saidnon-natural nucleic acid product is selected from antisense RNA,antisense DNA, sense RNA, sense DNA, a ribozyme and a protein bindingnucleic acid sequence.
 170. The composition of claim 169, wherein saidprotein binding nucleic acid sequence comprises a decoy that binds aprotein required for a viral assembly or viral replication.
 171. Thecomposition of claim 169, wherein said non-natural nucleic acid productcomprises antisense RNA or antisense DNA and said portion of thelocalizing entity (I) comprises a nuclear localization signallingsequence.
 172. The composition of claim 169, wherein said non-naturalnucleic acid product comprises antisense RNA or antisense DNA and saidportion of the localizing entity (I) comprises a cytoplasmiclocalization signalling sequence.
 173. The composition of claim 169,wherein said non-natural nucleic acid product comprises sense RNA orsense DNA and said portion of a localizing entity (I) comprises acytioplasmic localization signalling sequence.
 174. The composition ofclaim 161, wherein said nucleic acid component is selected from anucleic acid, a nucleic acid construct, a nucleic acid conjugate, avirus, a viral fragment, a viral vector, a viroid, a phage, a plasmid, aplasmid vector, a bacterium and a bacterial fragment, or a combinationof the foregoing.
 175. The composition of claim 174, wherein saidnucleic acid is selected from DNA, RNA, a DNA-RNA hybrid and a DNA-RNAchimera, or a combination of the foregoing.
 176. The composition ofclaim 174, wherein said nucleic acid is modified.
 177. The compositionof claim 161, wherein said cell is eukaryotic or prokaryotic.
 178. Thecomposition of claim 161, wherein the production of said nucleic acidproduct is mediated by a vector.
 179. The composition of claim 178,wherein said vector is selected from a viral vector, a phage vector anda plasmid vector, or a combination thereof.
 180. A cell containing thecomposition of claim
 161. 181. The cell of claim 180, wherein said cellis eukaryotic or prokaryotic.
 182. The cell of claim 180, wherein saidcomposition has been introduced ex vivo into said cell.
 183. The cell ofclaim 180, wherein said composition has been introduced in vivo intosaid cell.
 184. A biological system containing the cell of claim 180.185. The biological system of claim 184, wherein said system is selectedfrom an organism, an organ, a tissue and a culture, or a combinationthereof.
 186. A process for localizing a nucleic acid product in aeukaryotic cell, comprising: (a) providing a composition of mattercomprising a nucleic acid component which when present in a cellproduces a non-natural nucleic acid product, which product comprises:(i) a portion of a localizing entity, and (ii) a nucleic acid sequenceof interest; and (b) introducing said composition into said cell or intoa biological system containing said cell.
 187. The process of claim 186,wherein said portion of the localizing entity (I) is sufficient topermit localization of said nucleic acid product.
 188. The process ofclaim 186, wherein said nucleic acid product comprises antisense RNA orantisense DNA and said portion of a localizing entity (I) comprises anuclear localization signaling sequence.
 189. The process of claim 186,wherein said nucleic acid product comprises sense RNA or sense DNA andsaid portion of a localizing entity (i) comprises a nuclear localizationsignalling sequence.
 190. The process of claim 186, wherein said nucleicacid product comprises sense RNA or sense DNA and said portion of alocalizing entity (I) comprises a nuclear localization signallingsequence.
 191. The process of claim 186, wherein said nucleic acidproduct comprises snRNA.
 192. The process of claim 191, wherein saidsnRNA comprises U1 or U2 or both.
 193. The process of claim 186, whereinsaid composition is introduced ex vivo into said cell or into abiological system containing said cell.
 194. The process of claim 186,wherein said composition is introduced in vivo into said cell or into abiological system containing said cell.
 195. A nucleic acid componentwhich upon introduction into a cell is capable of producing more thanone specific nucleic acid sequence, each such specific sequence soproduced being substantially nonhomologous with each other and beingeither complementary with a specific portion of a single-strandednucleic acid of interest in a cell or capable of binding to a specificprotein of interest in a cell.
 196. The nucleic acid component of claim195, wherein said single-stranded nucleic acids of interest are part ofthe same polynucleotide sequence or part of different polynucleotidesequences.
 197. The nucleic acid component of claim 195, wherein saidsingle-stranded nucleic acids of interest comprise a viral sequence.198. The nucleic acid component of claim 195, wherein said component isderived or selected from a nucleic acid, a nucleic acid construct, anucleic acid conjugate, a virus, a viral fragment, a viral vector, aphage, a plasmid, a bacterium and a bacterial fragment, or a combinationof any of the foregoing.
 199. The nucleic acid component of claim 195,wherein said nucleic acid is selected from DNA, RNA and nucleic acidanalogs, or a combination thereof.
 200. The nucleic acid component ofclaim 199, wherein said DNA or RNA is modified.
 201. The nucleic acidcomponent of claim 195, comprising either more than one promoter or morethan one initiator, or both.
 202. The nucleic acid component of claim195, wherein each said specific nucleic acid sequence product is capableof being produced independently from either different promoters,different initiators, or a combination of both.
 203. The nucleic acidcomponent of claim 195, wherein said specific nucleic acid sequenceproducts are either complementary to a viral or cellular RNA, or bind toa viral or cellular protein, or or a combination of the foregoing. 204.The nucleic acid component of claim 203, wherein said complementaryspecific nucleic acid sequence products are capable of acting asantisense.
 205. The nucleic acid component of claim 204, wherein saidviral or cellular protein comprises a localizing protein or a decoyprotein.
 206. The nucleic acid component of claim 205, wherein saidlocalizing protein comprises a nuclear localizing protein or acytoplasmic localizing protein.
 207. The nucleic acid component of claim205, wherein said decoy protein binds a protein required for viralassembly or viral replication.
 208. The nucleic acid component of claim195, wherein said specific nucleic acid sequence products are selectedfrom antisense RNA, antisense DNA, a ribozyme and a protein bindingnucleic acid sequence, or a combination of any of the foregoing. 209.The nucleic acid component of claim 195, further comprising a means fordelivering said component to a cell containing the nucleic acid ofinterest or the specific protein of interest.
 210. A process forincreasing cellular resistance to a virus of interest, comprising: (a)providing: (i) transformed cells phenotypically resistant to said virus;and (ii) a reagent capable of binding to said virus or to avirus-specific site on said cells; and (b) administering said reagent toa biological system containing said cells to increase the resistance ofsaid cells to the virus of interest.
 211. The process of claim 210,wherein said biological system is selected from an organism, an organand a tissue, or a combination thereof.
 212. The process of claim 210,wherein said viral resistant cells (i) are eukaryotic or prokaryotic.213. The process of claim 210, wherein said viral resistant cells (i)comprise a nucleic acid sequence selected from antisense RNA, antisenseDNA, sense RNA, sense DNA, a ribozyme and a protein binding nucleic acidsequence, or a combination of the foregoing.
 214. The process of claim210, wherein said virus binding reagent (ii) is selected from anantibody, a virus binding protein, a cell receptor protein and an agentcapable of stimulating production of a virus binding protein, or acombination of the foregoing.
 215. The process of claim 214, whereinsaid antibody comprises a polyclonal or monoclonal antibody.
 216. Theprocess of claim 215, wherein said polyclonal or monoclonal antibody isspecific to an epitope of said virus of interest.
 217. The process ofclaim 214, wherein said virus binding protein comprises a CD4 receptor.218. The process of claim 214, wherein said cell receptor proteincomprises a gp24 protein.
 219. The process of claim 214, wherein saidproduction stimulating agent is selected from an immunological responseenhancing adjuvant and a viral antigen, or a combination of both. 220.The process of claim 210 wherein said reagent (ii) is administered invivo to said cells.
 221. The process of claim 210, wherein said reagent(ii) is administered ex vivo to said cells.
 222. The process of claim210, further comprising administering an additional viral resistanceenhancing agent (iii).
 223. The process of claim 222, wherein saidadditional viral resistance enhancing agent (iii) is selected from aprotease inhibitor, a nucleoside analog, or a combination thereof. 224.The process of claim 222, wherein said additional viral resistanceenhancing agent (iii) is administered before administering said bindingreagent (ii).
 225. The process of claim 222, wherein said additionalviral resistance enhancing agent (iii) is administered afteradministering said binding reagent (ii).
 226. The process of claim 222,wherein said additional viral resistance enhancing agent (iii) isadministered at about the same time that said binding reagent (ii) isadministered.
 227. A biological system with increased viral resistanceobtained by the process of claim
 210. 228. A biological system withincreased viral resistance obtained by the process of claim
 222. 229. Anucleic acid construct which when introduced into a cell produces anon-natural product, which product comprises two components: (i) abinding component capable of binding to a cellular component; and (ii) alocalization component capable of dislocating said cellular componentwhen bound to said product.
 230. The construct of claim 229, whereinsaid product comprises a protein or a nucleic acid, or a combination ofboth.
 231. The construct of claim 230, wherein said protein comprises anantibody.
 232. The construct of claim 231, wherein said antibodycomprises a polyclonal or monoclonal antibody.
 233. The construct ofclaim 232 wherein said polyclonal or monoclonal antibody is directed toa cellular component inside the cell.
 234. The construct of claim 229,wherein said cellular component is selected from a nucleic acid, aprotein, a virus, a phage, a product from another construct, ametabolite and an allosteric compound, or a combination of theforegoing.
 235. The construct of claim 234, wherein said protein isselected from a viral or non-viral enzyme, a gene suppressor, aphosphorylated protein, or a combination of the foregoing.
 236. Theconstruct of claim 235, wherein said phosphorylated protein comprises anoncogene.
 237. The construct of claim 229, wherein said bindingcomponent of said product is selected from a nucleic acid, a protein anda binding entity, or a combination thereof.
 238. The construct of claim229, wherein said nucleic acid comprises a sequence selected from acomplementary sequence to said cellular component and a sequence to anucleic acid binding protein, or a combination of both.
 239. Theconstruct of claim 237, wherein said protein is selected from anantibody, a receptor and a nucleic acid binding protein, or acombination of the foregoing.
 240. The construct of claim 237, whereinsaid binding entity is capable of binding metabolites.
 241. Theconstruct of claim 229, wherein said localization component is selectedfrom a nuclear localizing entity, a cytoplasmic localizing entity and acell membrane localizing entity, or a combination thereof.
 242. Theconstruct of claim 229, wherein said localization component comprises amember selected from a nucleic acid sequence, a nucleic acid structureand a peptide or oligopeptide, or a combination of the foregoing. 243.The construct of claim 242, wherein said nucleic acid structurecomprises a stem and loop structure.
 244. A process for dislocating acellular component in a cell, comprising: (I) providing: (a) a nucleicacid construct which when introduced into a cell produces a non-naturalproduct, which product comprises two components: (i) a binding componentcapable of binding to a cellular component; and (ii) a localizationcomponent capable of dislocating said cellular component when bound tosaid product; and (II) introducing said nucleic acid construct into acell of interest or a biological system containing said cell ofinterest.